Autologous platelet gel delivery system

ABSTRACT

The delivery system of the present invention is comprised of at least two chambers which store, individually, an activated blood component and an inactivated blood component. The first chamber comprises an activating agent and a filter which may be one in the same thus allowing the formed clot to be triturated thereby isolating the thrombin from the clot. The second chamber stores the inactivated blood component that when mixed with thrombin will produce a gel. The first or second chamber may further contain agents which are desired to be delivered to a specific site. The unique design of the delivery system allows for ease in operation of combining two agents at a specific time and place.

CROSS-REFERENCE TO OTHER APPLICATIONS

This patent application is a Continuation-in-Part Application of U.S.patent application Ser. No. 09/063,338, filed Apr. 20, 1998, now U.S.Pat. No. 6,444,228, currently on appeal, and entitled “Autologous FibrinSealant and Method for Making the Same,” which is a Continuation-in PartApplication of U.S. patent application Ser. No. 08/640,278, filed Apr.30, 1996, now abandoned, and entitled “Method for Making AutologousFibrin Sealant.”

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to novel methods, devices and apparatuses for thecentrifugal separation of a liquid into its components of varyingspecific gravities, and is more particularly directed toward a bloodseparation device useful, for example, in the separation of bloodcomponents for use in various therapeutic regimens.

2. Description of the State of Art

Centrifugation utilizes the principle that particles suspended insolution will assume a particular radial position within the centrifugerotor based upon their respective densities and will therefore separatewhen the centrifuge is rotated at an appropriate angular velocity for anappropriate period of time. Centrifugal liquid processing systems havefound applications in a wide variety of fields. For example,centrifugation is widely used in blood separation techniques to separateblood into its component parts, that is, red blood cells, platelets,white blood cells, and plasma.

The liquid portion of the blood, referred to as plasma, is aprotein-salt solution in which red and white blood cells and plateletsare suspended. Plasma, which is 90 percent water, constitutes about 55percent of the total blood volume. Plasma contains albumin (the chiefprotein constituent), fibrinogen (responsible, in part, for the clottingof blood), globulins (including antibodies) and other clotting proteins.Plasma serves a variety of functions, from maintaining a satisfactoryblood pressure and providing volume to supplying critical proteins forblood clotting and immunity. Plasma is obtained by separating the liquidportion of blood from the cells suspended therein.

Red blood cells (erythrocytes) are perhaps the most recognizablecomponent of whole blood. Red blood cells contain hemoglobin, a complexiron-containing protein that carries oxygen throughout the body whilegiving blood its red color. The percentage of blood volume composed ofred blood cells is called the “hematocrit.”

White blood cells (leukocytes) are responsible for protecting the bodyfrom invasion by foreign substances such as bacteria, fungi and viruses.Several types of white blood cells exist for this purpose, such asgranulocytes and macrophages which protect against infection bysurrounding and destroying invading bacteria and viruses, andlymphocytes which aid in the immune defense.

Platelets (thrombocytes) are very small cellular components of bloodthat help the clotting process by sticking to the lining of bloodvessels. Platelets are vital to life, because they help prevent bothmassive blood loss resulting from trauma and blood vessel leakage thatwould otherwise occur in the course of normal, day-to-day activity.

If whole blood is collected and prevented from clotting by the additionof an appropriate anticoagulant, it can be centrifuged into itscomponent parts. Centrifugation will result in the red blood cells,which weigh the most, packing to the most outer portion of the rotatingcontainer, while plasma, being the least dense will settle in thecentral portion of the rotating container. Separating the plasma and redblood cells is a thin white or grayish layer called the buffy coat. Thebuffy coat layer consists of the white blood cells and platelets, whichtogether make up about 1 percent of the total blood volume.

These blood components, discussed above, may be isolated and utilized ina wide range of diagnostic and therapeutic regimens. For example, redblood cells are routinely transfused into patients with chronic anemiaresulting from disorders such as kidney failure, malignancies, orgastrointestinal bleeding and those with acute blood loss resulting fromtrauma or surgery. The plasma component is typically frozen bycryoprecipitation and then slowly thawed to produce cryoprecipitatedantihemophiliac factor (AHF) which is rich in certain clotting factors,including Factor VIII, fibrinogen, von Willebrand factor and FactorXIII. Cryoprecipitated AHF is used to prevent or control bleeding inindividuals with hemophilia and von Willebrand's disease. Platelets andwhite blood cells, which are found in the buffy layer component, can beused to treat patients with abnormal platelet function(thrombocytopenia) and patients that are unresponsive to antibiotictherapy, respectively.

Various techniques and apparatus have been developed to facilitate thecollection of whole blood and the subsequent separation of therapeuticcomponents therefrom. Centrifugal systems, also referred to asblood-processing systems, generally fall into two categories,discontinuous-flow and continuous-flow devices.

In discontinuous-flow systems, whole blood from the donor or patientflows through a conduit into the rotor or bowl where componentseparation takes place. These systems employ a bowl-type rotor with arelatively large (typically 200 ml or more) volume that must be filledwith blood before any of the desired components can be harvested. Whenthe bowl is full, the drawing of fresh blood is stopped, the whole bloodis separated into its components by centrifugation, and the unwantedcomponents are returned to the donor or patient through the same conduitintermittently, in batches, rather than on a continuous basis. When thereturn has been completed, whole blood is again drawn from the donor orpatient, and a second cycle begins. This process continues until therequired amount of the desired component has been collected.

Discontinuous-flow systems have the advantage that the rotors arerelatively small in diameter but have the disadvantage that theextracorporeal volume (i.e., the amount of blood that is out of thedonor at any given time during the process) is large. This, in turn,makes it difficult or impossible to use discontinuous systems on peoplewhose size and weight will not permit the drawing of the amount of bloodrequired to fill the rotor. Discontinuous-flow devices are used for thecollection of platelets and/or plasma, and for the concentration andwashing of red blood cells. They are used to reconstitute previouslyfrozen red blood cells and to salvage red blood cells lostintraoperatively. Because the bowls in these systems are rigid and havea fixed volume, however, it is difficult to control the hematocrit ofthe final product, particularly if the amount of blood salvaged isinsufficient to fill the bowl with red blood cells.

One example of a discontinuous-flow system is disclosed by McMannis, etal., in his U.S. Pat. No. 5,316,540, and is a variable volume centrifugefor separating components of a fluid medium, comprising a centrifugethat is divided into upper and lower chambers by a flexible membrane,and a flexible processing container bag positioned in the upper chamberof the centrifuge. The McMannis, et al., system varies the volume of theupper chamber by pumping a hydraulic fluid into the lower chamber, whichin turn raises the membrane and squeezes the desired component out ofthe centrifuge. The McMannis, et al., system takes up a fairly largeamount of space, and its flexible pancake-shaped rotor is awkward tohandle. The McMannis, et al., system does not permit the fluid medium toflow into and out of the processing bag at the same time, nor does itpermit fluid medium to be pulled out of the processing bag by suction.

In continuous-flow systems, whole blood from the donor or patient alsoflows through one conduit into the spinning rotor where the componentsare separated. The component of interest is collected and the unwantedcomponents are returned to the donor through a second conduit on acontinuous basis as more whole blood is being drawn. Because the rate ofdrawing and the rate of return are substantially the same, theextracorporeal volume, or the amount of blood that is out of the donoror patient at any given time in the procedure, is relatively small.These systems typically employ a belt-type rotor, which has a relativelylarge diameter but a relatively small (typically 100 ml or less)processing volume. Although continuous-flow systems have the advantagethat the amount of blood that must be outside the donor or patient canbe relatively small, they have the disadvantage that the diameter of therotor is large. These systems are, as a consequence, large. Furthermore,they are complicated to set up and use. These devices are used almostexclusively for the collection of platelets.

Continuous-flow systems are comprised of rotatable and stationary partsthat are in fluid communication. Consequently, continuous-flow systemsutilize either rotary seals or a J-loop. A variety of types of rotarycentrifuge seals have been developed. Some examples of rotary centrifugeseals which have proven to be successful are described in U.S. Pat. Nos.3,409,203 and 3,565,330, issued to Latham. In these patents, rotaryseals are disclosed which are formed from a stationary rigid lowfriction member in contact with a moving rigid member to create adynamic seal, and an elastomeric member which provides a resilientstatic seal as well as a modest closing force between the surfaces ofthe dynamic seal.

Another rotary seal suitable for use in blood-processing centrifuges isdescribed in U.S. Pat. No. 3,801,142 issued to Jones, et al. In thisrotary seal, a pair of seal elements having confronting annularfluid-tight sealing surfaces of non-corrodible material are provided.These are maintained in a rotatable but fluid-tight relationship byaxial compression of a length of elastic tubing forming one of the fluidconnections to these seal elements.

Related types of systems which incorporate rotatable, disposable annularseparation chambers coupled via rotary seals to stationary tubingmembers are disclosed in U.S. Pat. Nos. 4,387,848; 4,094,461; 4,007,871;and 4,010,894.

One drawback present in the above-described continuous-flow systems hasbeen their use of a rotating seal or coupling element between thatportion of the system carried by the centrifuge rotor and that portionof the system which remains stationary. While such rotating seals haveprovided generally satisfactory performance, they have been expensive tomanufacture and have unnecessarily added to the cost of the flowsystems. Furthermore, such rotating seals introduce an additionalcomponent into the system which if defective can cause contamination ofthe blood being processed.

One flow system heretofore contemplated to overcome the problem of therotating seal utilizes a rotating carriage on which a single housing isrotatably mounted. An umbilical cable extending to the housing from astationary point imparts planetary motion to the housing and thusprevents the cable from twisting. To promote the desired ends of sterileprocessing and avoid the disadvantages of a discontinuous-flow systemwithin a single sealed system, a family of dual member centrifuges canbe used to effect cell separation. One example of this type ofcentrifuge is disclosed in U.S. Pat. No. RE 29,738 to Adams entitled“Apparatus for Providing Energy Communication Between a Moving and aStationary Terminal”. As is now well known, due to the characteristicsof such dual member centrifuges, it is possible to rotate a containercontaining a fluid, such as a unit of donated blood and to withdraw aseparated fluid component, such as plasma, into a stationary container,outside of the centrifuge without using rotating seals. Such containersystems utilize a J-loop and can be formed as closed, sterile transfersets.

The Adams patent discloses a centrifuge having an outer rotatable memberand an inner rotatable member. The inner member is positioned within androtatably supported by the outer member. The outer member rotates at onerotational velocity, usually called “one omega,” and the inner rotatablemember rotates at twice the rotational velocity of the outer housing or“two omega.” There is thus a one omega difference in rotational speed ofthe two members. For purposes of this document, the term “dual membercentrifuge” shall refer to centrifuges of the Adams type.

The dual member centrifuge of the Adams patent is particularlyadvantageous in that, as noted above, no seals are needed between thecontainer of fluid being rotated and the non-moving component collectioncontainers. The system of the Adams patent provides a way to processblood into components in a single, sealed, sterile system wherein wholeblood from a donor can be infused into the centrifuge while the twomembers of the centrifuge are being rotated.

An alternate to the apparatus of the Adams patent is illustrated in U.S.Pat. No. 4,056,224 to Lolachi entitled “Flow System for CentrifugalLiquid Processing Apparatus.” The system of the Lolachi patent includesa dual member centrifuge of the Adams type. The outer member of theLolachi centrifuge is rotated by a single electric motor which iscoupled to the internal rotatable housing by belts and shafts.

U.S. Pat. No. 4,108,353 to Brown entitled “Centrifugal Apparatus WithOppositely Positioned Rotational Support Means” discloses a centrifugestructure of the Adams type which includes two separate electricalmotors. One electric motor is coupled by a belt to the outer member androtates the outer member at a desired nominal rotational velocity. Thesecond motor is carried within the rotating exterior member and rotatesthe inner member at the desired higher velocity, twice that of theexterior member.

U.S. Pat. No. 4,109,855 to Brown, et al., entitled “Drive System ForCentrifugal Processing Apparatus” discloses yet another drive system.The system of the Brown, et al., patent has an outer shaft, affixed tothe outer member for rotating the outer member at a selected velocity.An inner shaft, coaxial with the outer shaft, is coupled to the innermember. The inner shaft rotates the inner member at twice the rotationalvelocity as the outer member. A similar system is disclosed in U.S. Pat.No. 4,109,854 to Brown entitled “Centrifugal Apparatus With OuterEnclosure”.

The continuous-flow systems described above are large and expensiveunits that are not intended to be portable. Further, they are also anorder of magnitude more expensive than a standard, multi-container bloodcollection set. There exists the need, therefore, for a centrifugalsystem for processing blood and other biological fluids that is compactand easy to use and that does not have the disadvantages of prior-artcontinuous-flow systems.

Whole blood that is to be separated into its components is commonlycollected into a flexible plastic donor bag, and the blood iscentrifuged to separate it into its components through a batch process.This is done by spinning the blood bag for a period of about 10 minutesin a large refrigerated centrifuge. The main blood constituents, i.e.,red blood cells, platelets and white cells, and plasma, havingsedimented and formed distinct layers, are then expressed sequentiallyby a manual extractor in multiple satellite bags attached to the primarybag.

More recently, automated extractors have been introduced in order tofacilitate the manipulation. Nevertheless, the whole process remainslaborious and requires the separation to occur within a certain timeframe to guarantee the quality of the blood components. This complicatesthe logistics, especially considering that most blood donations areperformed in decentralized locations where no batch processingcapabilities exist.

This method has been practiced since the widespread use of thedisposable plastic bags for collecting blood in the 1970's and has notevolved significantly since then. Some attempts have been made to applyhaemapheresis technology in whole blood donation. This techniqueconsists of drawing and extracting on-line one or more blood componentswhile a donation is performed, and returning the remaining constituentsto the donor. However, the complexity and costs of haemapheresis systemspreclude their use by transfusion centers for routine whole bloodcollection.

There have been various proposals for portable, disposable, centrifugalapparatus, usually with collapsible bags, for example as in U.S. Pat.Nos. 3,737,096, or 4,303,193 to Latham, Jr., or with a rigid walled bowlas in U.S. Pat. No. 4,889,524 to Fell, et al. These devices all have aminimum fixed holding volume which requires a minimum volume usually ofabout 250 ml to be processed before any components can be collected.

U.S. Pat. No. 5,316,540 to McMannis, et al., discloses a centrifugalprocessing apparatus, wherein the processing chamber is a flexibleprocessing bag which can be deformed to fill it with biological fluid orempty it by means of a membrane which forms part of the drive unit. Thebag comprises a single inlet/outlet tubing for the introduction andremoval of fluids to the bag, and consequently cannot be used in acontinual, on-line process. Moreover, the processing bag has a thedisadvantage of having 650 milliliter capacity, which makes theMcMannis, et al., device difficult to use as a blood processing device.

As discussed above, centrifuges are often used to separated blood intoits components for use in a variety of therapeutic regimens. One suchapplication is the preparation of a bioadhesive sealant. A bioadhesivesealant, also referred to as a fibrin glue, is a relatively newtechnological advance which attempts to duplicate the biological processof the final stage of blood coagulation. Clinical reports document theutility of fibrin glue in a variety of surgical fields, such as,cardiovascular, thoracic, transplantation, head and neck, oral,gastrointestinal, orthopedic, neurosurgical, and plastic surgery. At thetime of surgery, the two primary components comprising the fibrin glue,fibrinogen and thrombin, are mixed together to form a clot. The clot isapplied to the appropriate site, where it adheres to the necessarytissues, bone, or nerve within seconds, but is then slowly reabsorbed bythe body in approximately 10 days by fibrinolysis. Important features offibrin glue is its ability to: (1) achieve haemostasis at vascularanastomoses particularly in areas which are difficult to approach withsutures or where suture placement presents excessive risk; (2) controlbleeding from needle holes or arterial tears which cannot be controlledby suturing alone; and (3) obtain haemostasis in heparinized patients orthose with coagulopathy. See, Borst, H. G., et al., J Thorac.Cardiovasc. Surg., 84:548-553 (1982); Walterbusch, G. J, et al., Thorac.Cardiovasc. Surg., 30:234-23 5 (1982); and Wolner, F. J, et al., Thorac.Cardiovasc. Surg., 30:236-237 (1982).

Despite the effectiveness and successful use of fibrin glue by medicalpractitioners in Europe, neither fibrin glue nor its essentialcomponents fibrinogen and thrombin are widely used in the United States.In large part, this stems from the 1978 U.S. Food and DrugAdministration ban on the sale of commercially prepared fibrinogenconcentrate made from pooled donors because of the risk of transmissionof viral infection, in particular the hepatitis-causing viruses such asHBV and HCV (also known as non-A and non-B hepatitis virus). Inaddition, the more recent appearance of other lipid-enveloped virusessuch as HIV, associated with AIDS, cytomegalovirus (CMV), as well asEpstein-Barr virus and the herpes simplex viruses in fibrinogenpreparations makes it unlikely that there will be a change in thispolicy in the foreseeable future. For similar reasons, human thrombin isalso not currently authorized for human use in the United States. Bovinethrombin, which is licensed for human use in the United States isobtained from bovine sources which do not appear to carry significantrisks for HIV and hepatitis, although other bovine pathogens, such asbovine spongiform and encephalitis, may be present.

There have been a variety of methods developed for preparing fibringlue. For example, Rose, et al. in U.S. Pat. No. 4,627,879 discloses amethod of preparing a cryoprecipitated suspension containing fibrinogenand Factor XIII useful as a precursor in the preparation of a fibringlue which involves (a) freezing fresh frozen plasma from a single donorsuch as a human or other animal, e.g. a cow, sheep or pig, which hasbeen screened for blood transmitted diseases, e.g. one or more ofsyphilis, hepatitis or acquired immune deficiency syndrome, at about 80°C. for at least about 6 hours, preferably for at least about 12 hours;(b) raising the temperature of the frozen plasma, e.g. to between about0° C. and room temperature, so as to form a supernatant and acryoprecipitated suspension containing fibrinogen and Factor XIII; and(c) recovering the cryoprecipitated suspension. The fibrin glue is thenprepared by applying a defined volume of the cyroprecipitate suspensiondescribed above and applying a composition containing a sufficientamount of thrombin, e.g. human, bovine, ovine or porcine thrombin, tothe site so as to cause the fibrinogen in the suspension to be convertedto the fibrin glue which then solidifies in the form of a gel.

A second technique for preparing fibrin glue is disclosed by Marx in hisU.S. Pat. No. 5,607,694. Essentially, a cryoprecipitate as discussedpreviously serves as the source of the fibrinogen component and thenMarx adds thrombin and liposomes. A third method discussed by Berruyer,(M.,) et al., entitled “Immunization by bovine thrombin used with fibringlue during cardiovascular operations,” (J.) Thorac. Cardiovasc. Surg.,105(5):892-897 (1992)) discloses a fibrin glue prepared by mixing bovinethrombin not only with human coagulant proteins, such as fibrinogen,fibronectin, Factor XIII, and plasminogen, but also with bovineaprotinin and calcium chloride.

The above patents by Rose, et al., and Marx, and the technical paper byBerruyer, et al. each disclose methods for preparing fibrin sealants;however, each of these methods suffer disadvantages associated with theuse of bovine thrombin as the activating agent. A serious and lifethreatening consequence associated with the use of fibrin gluescomprising bovine thrombin is that patients have been reported to have ableeding diathesis after receiving topical bovine thrombin. Thiscomplication occurs when patients develop antibodies to the bovinefactor V in the relatively impure bovine thrombin preparations. Theseantibodies cross-react with human factor V, thereby causing a factor Vdeficiency that can be sufficiently severe to induce bleeding and evendeath. See, Rapaport, S. I., et al., Am. (J.) Clin. Pathol., 97:84-91(1992); Berruyer, M., et al., J. Thorac. Cardiovasc. Surg., 105:892-897(1993); Zehnder, J., et al., Blood, 76(10):2011-2016 (1990); Muntean,W., et al., Acta Paediatr., 83:84-7 (1994); Christine, R. J., et al.,Surgery, 127:708-710 (1997).

Further disadvantages associated with the methods disclosed by Marx andRose, et al. are that the cryoprecipitate preparations require a largetime and monetary commitment to prepare. Furthermore, great care must betaken to assure the absence of any viral contaminants.

A further disadvantage associated with the methods previously disclosedis that while human thrombin is contemplated for use as an activator,human thrombin is not available for clinical use and there is noevidence that patients will not have an antigenic response to humanthrombin. By analogy, recombinant human factor VIII has been shown toproduce antigenic responses in hemophiliacs. See, Biasi, R. de.,Thrombosis and Haemostasis, 71(5):544-547 (1994). Consequently, untilmore clinical studies are performed on the effect of human recombinantthrombin one cannot merely assume that the use of recombinant humanthrombin would obviate the antigenic problems associated with bovinethrombin. A second difficulty with thrombin is that it is autocatalytic,that is, it tends to self-destruct, making handling and prolongedstorage a problem.

Finally, as discussed above, fibrin glue is comprised primarily offibrinogen and thrombin thus lacking an appreciable quantity ofplatelets. Platelets contain growth factors and healing factors whichare assumed to be more prevalent in a platelet concentrate. Moreover,platelets aid in acceleration of the clotting process.

There is still a need, therefore, for a centrifugal system forprocessing blood and other biological fluids, that is compact and easyto use and that does not have the disadvantages of prior-artcontinuous-flow systems and furthermore there exists a need for aconvenient and practical method for preparing a platelet gel compositionwherein the resulting platelet gel poses a zero risk of diseasetransmission and a zero risk of causing an adverse physiologicalreaction.

There is also a widespread need for a system that, during bloodcollection, will automatically separate the different components ofwhole blood that are differentiable in density and size, with a simple,low cost, disposable unit.

There is further a need for a centrifugal cell processing system whereinmultiple batches of cells can be simultaneously and efficientlyprocessed without the use of rotational coupling elements.

There is yet a further need for a platelet concentrate that aids inincreasing the rate of fibrin clot formation, thereby facilitatinghaemostasis.

Preferably the apparatus will be essentially self-contained. Preferably,the equipment needed to practice the method will be relativelyinexpensive and the blood contacting set will be disposable each timethe whole blood has been separated.

SUMMARY OF THE INVENTION

Accordingly, one object of this invention is to provide a method andapparatus for the separation of components suspended or dissolved in afluid medium by centrifugation. More specifically, one object of thisinvention is to provide a method for the separation and isolation of oneor more whole blood components, such as platelet rich plasma, whiteblood cells and platelet poor plasma, from anticoagulated whole blood bycentrifugation, wherein the components are isolated while the centrifugeis rotating.

Another object of this invention is to utilize the isolated cellcomponents in a therapeutic regimen.

Another object of this invention is to provide an apparatus for theseparation of whole blood components, wherein the apparatus contains acentrifuge bag that provides for simultaneous addition of whole bloodfrom a source container and the withdrawal of a specific blood componentduring centrifugation.

Another object of this invention is to provide disposable, single-usecentrifuge bags for holding whole blood during the separation ofcomponents of the whole blood by centrifugation, wherein the bag isadapted for use in a portable, point-of-use centrifuge.

Another object of this invention is to provide a portable centrifugecontaining a disposable centrifuge bag that maximizes the amount of apredetermined blood fraction that can be harvested from an aliquot ofblood that is of greater volume than the capacity of the disposablecentrifuge bag.

To achieve the foregoing and other objects and in accordance with thepurposes of the present invention, as embodied and broadly describedtherein, one embodiment of this invention comprises a flexible,disposable centrifuge bag adapted to be rotated about an axis,comprising:

-   -   a) one or more tubes, and    -   b) upper and lower flexible sheets, each sheet having a doughnut        shaped configuration, an inner perimeter defining a central core        and an outer perimeter, wherein the upper and lower sheets are        superimposed and completely sealed together at their outer        perimeters, and wherein the tubes are sandwiched between the        upper and lower sheets and extend from the central core toward        the outer perimeter, such that when the upper and lower sheets        are sealed at the inner perimeter the tubes are sealed between        the upper and lower sheets at the inner perimeter and are in        fluid communication with the environment inside and outside the        centrifuge bag. The one or more tubes are fluidly connected to        an umbilical cable comprising one or more lumen equal to the        number of tubes of the centrifuge bag.

To further achieve the foregoing and other objects of this invention,another embodiment of the present invention comprises a rigid moldedcontainer adapted to be rotated about an axis, comprising a rigid,annular body having an axial core that is closed at the top end andopened at the bottom end. The rigid molded container further comprisesan interior collection chamber for receiving and holding a fluid mediumto be centrifuged, the chamber having an outer perimeter, an innerperimeter, and a generally off-centered “figure eight” shapedcross-sectional area. The rigid molded container further comprises afirst channel which extends radially from the core and is in fluidcommunication with a point near the outer perimeter of the chamber, anda second channel which extends radially from the core and is in fluidcommunication with an area near the narrow portion or “neck” of thefigure eight-shaped chamber. The first and second channels thus providefluid communication with the environment inside and outside the interiorcollection chamber. The first and second channels are fluidly connectedto a dual lumen tubing having an inlet lumen and an outlet lumen.

To further achieve the foregoing and other objects of this invention,another embodiment of the present invention is an apparatus and methodfor separating components contained in a fluid medium. Moreparticularly, the present invention utilizes the principles ofcentrifugation to allow for the separation of whole blood into fractionssuch as platelet rich plasma and platelet poor plasma. In one aspect ofthe present invention, the above-described separation of the componentsis provided by utilizing a rotatable centrifuge motor comprising a basehaving a central column and a disposable centrifuge bag having a centralcore and which is positionable within the centrifuge motor and rotatabletherewith. The disposable centrifuge bag, which holds the whole bloodduring centrifugation, further comprises an inlet tube for introducingthe whole blood to the centrifuge bag, and an outlet tube for removingthe desired blood fraction from the centrifuge bag. The inlet and outlettubes are in fluid communication with a dual lumen tubing. Thecentrifuge bag is removably fixed within the centrifuge rotor byinserting the raised column through the bag center core and securingwith the cover. During the rotation of the centrifuge, components of thewhole blood will assume a radial, horizontal position within thecentrifuge bag based upon a density of such components, and thus thefluid medium components will be separated from other components havingdifferent densities.

Once a desired degree of separation of whole blood has been achieved,the present invention provides for the specific removal of the desiredfraction within one or more of the regions from the centrifuge bagthrough the outlet tube during continued rotation of the centrifuge,thereby allowing for on-line removal of the desired fraction. Additionalaliquots may be added to the centrifuge bag via the inlet tubesimultaneously or after the desired component has been harvested. In oneembodiment, the centrifuge bag is a flexible, transparent, generallyflat doughnut-shaped bag. In another embodiment, the centrifuge bag is arigid, transparent container having an interior chamber for receivingand holding the fluid medium during centrifugation, the interior chamberhaving a generally off-centered figure eight cross-sectionalconfiguration.

Another aspect of the present invention comprises a disposablecentrifuge bag having an inlet tube and an outlet tube, wherein theoutlet tube is fluidly connected with a bent fitting.

Another aspect of the present invention comprises a centrifuge rotor forholding a centrifuge bag, the rotor comprising a base and a cover, thebase further having a first grooved, raised center column and the coverhaving a second grooved, raised center column. The centrifuge bag is aflexible, doughnut-shaped bag comprising inlet and outlet tubes in fluidcommunication with the environment inside and outside the centrifugebag, wherein the tubes are seated in the base and cover column groovesto hold the centrifuge bag in a fixed position relative to the base andcover, such that the bag does not spin independently of the base andcover but rather spins concurrently and at the same rate of rotation asthe base and cover.

Another aspect of the present invention comprises a centrifuge rotor forholding a centrifuge bag, the rotor comprising a base and a cover forsecuring a centrifuge bag therebetween, the centrifuge cover furthercomprising one or more concentric indicator circles that are spaced fromthe center of the cover or the base to aid the operator in visualizingthe distal ends of these tubes.

Another aspect of the present invention for the separation of componentsof a fluid medium (e.g., whole blood) utilizes a centrifuge rotorcomprising an interior chamber having a complex configuration, whereinthe chamber holds a flexible, doughnut-shaped centrifuge bag forretaining the fluid medium during centrifugation. The centrifuge rotoris defined by a base having a lower chamber, and a cover having an upperchamber. When the cover is superimposed on the base, the upper and lowerchambers define the annular interior chamber of the rotor. The interiorrotor chamber has a generally off-centered figure eight-shapedcross-sectional configuration specifically designed to maximize thecollection of the desired component (e.g., platelet rich plasma) bycentrifugation of a fluid medium (e.g., anticoagulated whole blood). Thecentrifuge bag is formed from a substantially flexible material, suchthat the profile of the centrifuge bag during centrifugation is thusdetermined at least in part by the volume of the fluid medium containedtherein. When the centrifuge bag is filled to maximum capacity, itassumes the configuration of the interior of the rotor chamber.

Another aspect of this invention comprises a method for on-lineharvesting of a predetermined component of a fluid medium. Oneembodiment of the present invention utilizes a centrifuge and adisposable centrifuge bag for containing the fluid medium duringseparation and which is positionable within the centrifuge, thecentrifuge bag further comprising at least one inlet tube and at leastone outlet tube. The centrifuge includes a centrifuge rotor having abase portion, a cover, and an outer rim. The base portion and the coverdefine the interior of the centrifuge rotor, which is separated intoupper and lower chambers. The disposable centrifuge bag is positionablehorizontally within the lower chamber and may be appropriately securedto the centrifuge base by the cover. The centrifuge bag is fluidlyconnected via a dual lumen tubing to a source (e.g., to a containercomprising anticoagulated autologous whole blood) and collectioncontainer (e.g., for receiving platelet rich plasma or some othercomponent that will then be further processed). The dual lumen tubingcomprises an inlet lumen fluidly connected to the inlet tube of thecentrifuge bag and an outlet lumen fluidly connected to the outlet tubeof the centrifuge bag. The centrifuge bag is substantially annularrelative to the rotational axis of the centrifuge. When the centrifugebag is positioned within the centrifuge rotor and appropriately securedthereto to allow for simultaneous rotation, the fluid medium may beprovided to the centrifuge bag via the inlet lumen of the tubing duringrotation of the centrifuge. The components of the bag assume radial,horizontal positions base based on their densities. When a desireddegree of separation has been achieved, the desired fraction may beremoved from the centrifuge bag via the outlet lumen during continuedrotation of the centrifuge. The position of the fraction to be harvestedmay be shifted into the area of the outlet tube as needed, either bywithdrawing components that are positioned near the outer perimeterthrough the inlet tube, or by adding additional aliquots of the fluidmedium to the bag. In one embodiment of this method, the bag is aflexible, transparent doughnut-shaped bag. In another embodiment of thismethod, the bag is a rigid, transparent bag comprising an interiorchamber having an off-centered, figure eight cross-sectionalconfiguration.

It is yet another object of the invention to provide a centrifugalliquid processing system that may be automated.

It is yet another object of the present invention to provide acentrifuge having an internal lead drive mechanism allowing for acompact size.

A further object of the present invention is to provide for a method anddevice for the production and isolation of thrombin for all medicaluses.

It is yet another object of this invention to provide a method forpreparing a completely autologous platelet gel.

Another object of the present invention is to provide an autologousplatelet gel wherein the risks associated with the use of bovine andrecombinant human thrombin are eliminated.

A further object of the present invention is to provide an autologousplatelet gel for any application.

It is a further object of the present invention to provide cellularcomponents to be used in medical applications.

Additional objects, advantages, and novel features of this inventionshall be set forth in part in the description and examples that follow,and in part will become apparent to those skilled in the art uponexamination of the following or may be learned by the practice of theinvention. The objects and the advantages of the invention may berealized and attained by means of the instrumentalities and incombinations particularly pointed out in the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and form a part ofthe specifications, illustrate the preferred embodiments of the presentinvention, and together with the description serve to explain theprinciples of the invention.

In the Drawings:

FIG. 1 is a perspective view illustrating one embodiment of thecontinuous-flow centrifugal processing system of the present inventionillustrating a centrifuge and side-mounted motor positioned within aprotective housing or enclosure of the invention.

FIG. 2 is an exploded side view of the centrifuge and the side-mountedmotor of the centrifugal processing system of FIG. 1 illustrating theindividual components of the centrifuge.

FIG. 3 is a partial perspective view of the lower case assembly of thedrive shaft assembly of FIG. 2.

FIG. 4 is an exploded side view of the lower case assembly of FIG. 3.

FIG. 5 is an exploded perspective view of the components of the lowercase assembly of FIG. 3.

FIG. 6 is a top view of the lower bearing assembly which is positionedwithin the lower case assembly of FIG. 3.

FIG. 7 is a perspective view of the lower bearing assembly of FIG. 6.

FIG. 8 is an exploded side view of the lower bearing assembly of FIGS. 6and 7.

FIG. 9 is a perspective view of the receiving tube guide of thecentrifuge of FIG. 2.

FIG. 10 is an exploded, perspective view of a gear of the mid-shaft gearassembly of FIG. 2.

FIG. 11 is a perspective view of the gear of FIG. 10 as it appearsassembled.

FIG. 12 is an exploded, perspective view of the top bearing assembly ofthe centrifuge of FIG. 2.

FIG. 13 is a perspective view of the top case shell of the top bearingassembly of FIG. 12.

FIG. 14 is a perspective view of the centrifuge of the present inventionshown in FIG. 1, having a quarter section cut away along lines 14—14 ofFIG. 1.

FIG. 15 is a perspective view of one embodiment of a centrifuge rotorbase.

FIG. 16 is a perspective view of one embodiment of a centrifuge rotorcover.

FIG. 17 is a side cross-sectional view of one embodiment of a rotor ofthis invention taken along view lines 17 of FIG. 14 for holding adisposable centrifuge bag, showing a dual lumen tubing connected to thebag.

FIG. 18 is a side cross-sectional view of one embodiment of a rotor ofthis invention taken along view lines 18 of FIG. 1 for holding adisposable centrifuge bag, showing the grooved columns of the base andcover.

FIG. 19 is an enlarged perspective view similar to FIG. 1 illustratingan alternate embodiment of a centrifuge driven by a side-mounted motor(with only the external drive belt shown).

FIG. 20 is a cutaway side view of the centrifuge of FIG. 19 illustratingthe internal pulley drive system utilized to achieve a desired driveratio and illustrating the rotor base configured for receiving acentrifuge bag.

FIG. 21 is a cutaway side view similar to FIG. 20 with the rotor baseremoved to better illustrate the top pulley and the location of bothidler pulleys relative to the installed internal drive belt.

FIG. 22 is a sectional view of the centrifuge of FIG. 20 furtherillustrating the internal pulley drive system an showing the routing ofthe centrifuge tube (or umbilical cable).

FIG. 23 is a top view of a further alternate centrifuge similar to thecentrifuge of FIG. 19 but including internal, separate bearing members(illustrated as four cam followers) that allows the inclusion of guideshaft to be cut through portions of the centrifuge for positioning ofthe centrifuge tube (or umbilical cable).

FIG. 24 is a perspective view similar to FIG. 19 illustrating thecentrifuge embodiment of FIG. 23 further illustrating the guide slot andshowing that the centrifuge can be driven by an external drive belt.

FIG. 25 is a top view of a flexible, disposable centrifuge bag of thisinvention.

FIG. 26 is a perspective view of a flexible, disposable centrifuge bagof this invention.

FIGS. 27, 28, 29, and 30 are illustrations of bent fittings of thisinvention having “T” shaped, “curved T” shaped, “L” shaped, and “J”shaped configurations, respectively.

FIG. 31 is an illustration of an inlet and/or outlet tube of thisinvention.

FIG. 32 is a top view of a disposable centrifuge bag of this inventionafter the centrifugation of whole blood, showing the separated bloodcomponents.

FIGS. 33-39 are schematic illustrations of one method of this inventionfor separating whole blood components using a disposable centrifuge bagof this invention.

FIG. 40 is a top view of an alternate embodiment of a disposablecentrifuge bag of the present invention having inner and outer chambers.

FIG. 41 is a top view of the disposable centrifuge bag shown in FIG. 34illustrating movement of the red blood cell layer from the outerperimeter toward the inner perimeter.

FIG. 42 is a bottom view of an alternate embodiment of a disposablecentrifuge bag of the present invention having inner and outer chambersin fluid communication with outlet and inlet ports.

FIG. 43 is a side cross-sectional view of a rigid disposable centrifugebag of this invention.

FIG. 44 is a schematic illustration of separated blood componentscontained in a centrifuge bag having an elliptical cross-sectional viewof the centrifuge bag shown in FIG. 43.

FIG. 45 is a side cross-sectional view of a rigid disposable centrifugebag of this invention.

FIG. 46 is a schematic illustration of the surface areas and variousdimensions of the figure eight configuration as shown in FIG. 45.

FIG. 47 is a schematic illustration of separated blood componentscontained in a centrifuge bag having a figure eight side cross-sectionalconfiguration.

FIG. 48 is a side cross-sectional view of an alternative embodiment ofan assembled centrifuge rotor of this invention comprising the rotorcover of FIG. 49 and the rotor base of FIG. 50.

FIG. 49 is a side cross-sectional view of an alternative embodiment of arotor cover of this invention.

FIG. 50 is a side cross-sectional view of an alternative embodiment of arotor base of this invention.

FIG. 51 is a perspective view of the rotor base of FIG. 50.

FIG. 52 is a perspective view of the rotor cover of FIG. 49.

FIG. 53 is a block diagram illustrating the components of a centrifugalprocessing system of the present invention.

FIG. 54 is a graph illustrating the timing and relationship oftransmission of control signals and receipt of feedback signals duringoperation of one embodiment of the automated centrifugal processingsystem of FIG. 53.

FIG. 55 is a side view of an alternative embodiment of the automatedcentrifugal processing system of FIG. 53 showing a centrifuge having arotor wherein the reservoir extends over the outer diameter of thecentrifuge portion that facilitates use of an externally-positionedsensor assembly.

FIG. 56 is a side view of a further alternative embodiment of theexternal sensor assembly feature of the centrifugal processing system ofthe invention without an extended rotor and illustrating the positioningof a reflector within the centrifuge.

FIG. 57 is a side view of yet another embodiment of the external sensorassembly feature of the centrifugal processing system of the inventionillustrating a single radiant energy source and detector device.

FIG. 58 is a block diagram of a an automated centrifugal processingsystem, similar to the embodiment of FIG. 47, including componentsforming a temperature control system for controlling temperatures ofseparated and processed products.

FIG. 59 is a perspective view of components of the temperature controlsystem of FIG. 58.

FIG. 60 is schematic and sectional view of the dispenser of the presentinvention.

FIG. 61 is a flow diagram representing the method for isolating plateletrich plasma and platelet poor plasma for use in preparing a platelet gelof the present invention.

FIG. 62 is a flow diagram representing the final portion of the methodfor preparing a platelet gel of the present invention using plateletrich plasma as a starting material.

FIG. 63 is a flow diagram representing the final portion of the methodfor preparing a platelet gel of the present invention using plateletpoor plasma as a starting material.

FIG. 64 is a graphic representation of the effect that theserum-to-plasma ratio has on clotting times.

FIG. 65 graphically represents the effect of calcium addition on theclotting times of platelet rich plasma and platelet poor plasma.

FIG. 66 is a graphic representation of the relationship between clottingtime and actual gel time using blood drawn from a donor.

FIG. 67 is a graphic representation of the relationship between clottingtime and actual gel time using blood drawn from a donor.

FIG. 68 graphically represents the effect of calcium addition onclotting times and gel times using blood drawn from a donor.

FIG. 69 graphically represents the effect of calcium addition onclotting times and gel times using blood drawn from a donor.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The centrifugal processing system 10 of the present invention is bestshown in FIG. 1 having a stationary base 12, a centrifuge 20 rotatablymounted to the stationary base 12 for rotation about a predeterminedaxis A, a rotor 202 for receiving a disposable bag (not shown) designedfor continuous-flow. As illustrated, the centrifugal processing system10 includes a protective enclosure 11 comprising the main table plate orstationary base 12, side walls 13, and a removable lid 15 made of clearor opaque plastic or other suitable materials to provide structuralsupport for components of the centrifugal processing system 10, toprovide safety by enclosing moving parts, and to provide a portablecentrifugal processing system 10. The centrifugal processing system 10further includes a clamp 22 mounted over an opening (not shown) in thelid 15. Clamp 22 secures at a point at or proximately to axis A withoutpinching off the flow of fluid that travels through umbilical cable 228.A side mounted motor 24 is provided and connected to the centrifuge 20by way of a drive belt 26 for rotating the drive shaft assembly 28 (seeFIG. 2) and the interconnected and driven rotor assembly 200 in the samerotational direction with a speed ratio selected to control binding ofumbilical cable 228 during operation of the system, such as a speedratio of 2:1 (i.e., the rotor assembly 200 rotates twice for eachrotation of the drive shaft assembly 28). The present invention isfurther directed toward a dispensing device 902, best shown in FIG. 60for the withdrawal and manipulation of specific blood components forvarious therapeutic regimens, such as but not limited to the productionof platelet rich plasma, platelet poor plasma, and white blood cellswhich may be used for the production of autologous thrombin andautologous platelet gels.

Referring now to FIG. 2, the continuous-flow centrifugal processingsystem 10 comprises a centrifuge 20 to which a rotor 202 is removably ornon-removably attached. The design of centrifuge 20 and itsself-contained mid-shaft gear assembly 108 (comprised of gears 110,110′, 131, and 74) is a key component of the invention thereby allowingfor the compact size of the entire centrifugal processing system 10 andproviding for a desired speed ratio between the drive shaft assembly 28and the rotor assembly 200.

The centrifuge 20 is assembled, as best seen in FIG. 2, by inserting thelower bearing assembly 66 into lower case shell 32 thus resulting inlower case assembly 30. Cable guide 102 and gears 110 and 110′ are thenpositioned within lower case assembly 30, as will be discussed in moredetail below, so that gears 110 and 110′ are moveably of engaged withlower bearing assembly 66. Upper bearing assembly 130 is then insertedwithin top case shell 126 thus resulting in bearing assembly 124 whichis then mated to lower case assembly 30, such that gears 110 and 110′are also moveably engaged with upper bearing assembly 130, and held inplace by fasteners 29. Lower bearing assembly 66 is journaled tostationary base or main table plate 12 by screws 14, thus allowingcentrifuge 20 to rotate along an axis A, perpendicular to main tableplate 12 (as shown in FIG. 1).

Referring now to FIGS. 3, 4, and 5, the lower case assembly 30 ispreferably, but not necessarily, machined or molded from a metalmaterial and includes a lower case shell 32, timing belt ring 46, timingbelt flange 50, and bearing 62 (e.g., ball bearings and the like). Lowercase shell 32 includes an elongated main body 40 with a smaller diameterneck portion 36 extending from one end of the main body 40 for receivingtiming belt ring 46 and timing belt flange 50. The larger diameter mainbody 40 terminates into the neck portion 36 thereby forming an externalshoulder 38 having a bearing surface 42 for timing belt ring 46. Timingbelt ring 46 and timing belt flange 50, as best seen in FIG. 5, haveinner diameters that are slightly larger than the outer diameter of neckportion 36 allowing both to fit over neck portion 36. Shoulder 38further contains at least one and preferably four internally threadholes 44 that align with hole guides 48 and 52 in timing belt ring 46and timing belt flange 50, respectively (shown in FIG. 5). Consequently,when assembled, screws 54 are received by hole guides 52 and 48 and arethreaded into thread holes 44 thus securing timing belt 46 and timingbelt flange 50 onto neck portion 36. Lower case shell 32 also has anaxial or sleeve bore 56 extending there through, and an internalshoulder 58, the upper surface 60 of which is in approximately the samehorizontal plane as external shoulder 38. Bearing 62 (shown in FIG. 4)is press fit concentrically into sleeve bore 56 so that it sits flushwith upper surface 60. Internal shoulder 58 also has a lower weightbearing surface 64 which seats on the upper surface 68 of lower bearingassembly 66, shown in FIGS. 6-8.

Lower bearing assembly 66 comprises a lower gear insert 70, ballbearings 84, gear 74 and spring pins 76 and 76′. As will become clear,the gear 74 may be of any suitable gear design for transferring an inputrotation rate to a mating or contacting gear, such as the gears 110,110′ of the mid-shaft gear assembly 108, with a size and tooth numberselected to provide a desired gear train or speed ratio when combinedwith contacting gears. For example, the gear 74 may be configured as astraight or spiral bevel gear, a helical gear, a worm gear, a hypoidgear, and the like out of any suitable material. In a preferredembodiment, the gear 74 is a spiral gear to provide a smooth toothaction at the operational speeds of the centrifugal processing system10. The upper surface 68 of lower gear insert 70 comprises an axiallypositioned sleeve 72, which receives and holds gear 74. Gear 74 ispreferably retained within sleeve 72 by the use of at least one andpreferably two spring pins 76 and 76′ which are positioned within springpin holes 73 and 73′ extending horizontally through lower gear insert 70into sleeve 72. Thus, when gear 74 having spring pin receptacles 77 and77′ is inserted into sleeve 72 the spring pins 76 and 76′ enter thecorresponding receptacles 77 and 77′ thus holding the gear 74 in place.Of course, other assembly techniques may be used to position and retaingear 74 within the lower gear assembly 66 and such techniques areconsidered within the breadth of this disclosure. For example, gear 74may be held in sleeve 72 by a number of other methods, such as, but notlimited to being press fit or frictionally fit, or alternatively gear 74and lower gear insert 70 may be molded from a unitary body.

The base 78 of lower gear insert 70 has a slightly larger diameter thanupper body 80 of lower gear insert 70 as a result of a slight flare.This slight flare produces shoulder 82 upon which ball bearing 84 isseated. Once assembled lower bearing assembly 66 is received by sleevebore 56 extending through neck portion 36 of lower case shell 32. Aretaining ring 86 is then inserted into the annular space produced bythe difference of the outer diameter of the lower bearing assembly 66and the inner diameter of sleeve bore 56 above ball bearings 84. Asecond retaining ring 87 (shown in FIG. 2) is also inserted into theannular space produced by the difference between the outer diameter ofthe lower bearing assembly 66 and the inner diameter of sleeve bore 56below ball bearing 84, thereby securing lower gear insert 70 withinlower case shell 32. Consequently, ball bearings 62 and 84 are securedby retaining rings 86 and 87, respectively, resulting in lower caseshell 32 being journaled for rotation about lower bearing assembly 66but fixed against longitudinal and transverse movement thereon.Therefore, when assembled lower bearing assembly 66 is mounted tostationary base 12, by securing screws 14 into threaded holes 79 locatedin the base 78. Lower case shell 32 is thus able to freely rotate aboutstationary lower bearing assembly 66 when the drive belt 26 is engaged.

Referring now to FIG. 5, extending from the opposite end of neck portion36 on lower case shell 32 are a number of protrusions or fingers 88, 90,92, and 94. Positioned between protrusions 88 and 90, and betweenprotrusions 92 and 94 are recessed slots 96 and 98, respectively, forreceiving tube guide 102 (FIG. 9). The function of tube guide 102 willbe discussed in further detail below, but in short it guides umbilicalcable 228 connected to centrifuge bag 226 through the mid-shaft gearassembly 108 and out of the centrifuge 20.

Positioned between protrusions 90 and 92, and between protrusions 88 and94 are recessed slots 104 and 106, respectively, for receiving gears 110and 110′ of mid-shaft gear assembly 108 (FIG. 2). The gears 110 and 110′are preferably configured to provide mating contact with the gear 74 andto produce a desired, overall gear train ratio within the centrifuge 20.In this regard, the gears 110 and 110′ are preferably selected to have asimilar configuration (e.g., size, tooth number, and the like) as thegear 74, such as a spiral gear design. As illustrated in FIGS. 2 and 14mid-shaft gear assembly 108 comprises a pair of gears 110 and 110′engaged with gears 74 and 131. While the construction of gears and gearcombinations is well known to one skilled in the mechanical arts, abrief description is disclosed briefly herein.

FIG. 10 illustrates an exploded view depicting the assembly of gear 110,and FIG. 11 is a perspective view of the gear 110 of FIG. 10 as itappears assembled. Gear 110′ is constructed in the same manner. Gear 111is locked onto mid-gear shaft 112 using key stock 114 and externalretaining ring 116. Ball bearing 118 is then attached to mid gear shaft112 using a flat washer 120 and cap screw 122. Recessed slots 104 and106 of lower case shell 32 then receive ball bearing 118 and 118′ (notshown). In an alternate embodiment ball bearing 118 can be replaced bybushings (not shown). When assembled, gears 110 and 110′ make contactwith the lower gear 74 (see FIGS. 2 and 14) to provide contact surfacesfor transferring a force from the stationary gear 74 to the gears 110and 110′ to cause the gears 110 and 110′ to rotate at a predeterminedrate that creates a desired output rotation rate for the driven rotorassembly 200. The rotor assembly 200 is driven by the drive shaftassembly 28 which is rotated by the drive motor 24 at an input rotationrate or speed, and in a preferred embodiment, the drive shaft assembly28 through the use of the gears 110 and 110′ is configured to rotate therotor assembly 200 at an output rotation rate that is twice the inputrotation rate (i.e., the ratio of the output rotation rate to the inputrotation rate is 2:1). This ratio is achieved in the illustratedembodiment by locking the gears 110 and 110′ located within the driveshaft assembly 28 to rotate about the centrifuge center axis, A, withthe lower case shell 32 which is rotated by the drive motor 24. Thegears 110 and 110′ also contact the stationary gear 74 which forces thegears 110, 110′ to rotate about their rotation axes which are traverseto the centrifuge center axis, A, and as illustrated, the rotation axesof the gears 110, 110′ coincide. By rotating with the lower case shell32 and rotating about the gear rotation axes, the gears 110, 110′ areable to provide the desired input to output rotation rate of 2:1 to therotor assembly 200.

In this regard, gears 110 and 110′ and tube guide 102 are locked intoposition by attaching top bearing assembly 124 to lower case assembly30. Top bearing assembly 124 (as shown in FIG. 12) comprises top caseshell 126, ball bearing 128, and an upper bearing 130. Top case shell126, as best seen in FIGS. 12 and 13, comprises an upper surface 132, alower lip 134 and a central or axial bore 136 there through. Uppersurface 132 slightly overhangs axial bore 136 resulting in a shoulder138 having a lower surface 140 (shown in FIG. 13). Lower lip 134 is areverse image of upper lip 100 on lower case shell 32 (shown in FIG. 5).

Upper bearing assembly 130 (FIG. 12) comprises an upper surface 133 anda lower surface 135 wherein the upper surface 133 has a means forreceiving a rotor 202. On the lower surface 135 a concentricallypositioned column 137 protrudes radially outward perpendicular to lowersurface 135. Upper bearing assembly 130 further comprises an axiallypositioned bore 139 that traverses column 137 and upper surface 133 andreceives upper gear insert 131. Upper gear insert 131 also contains anaxial bore 142 and thus when positioned concentrically within column 137axial bores 139 and 142 allow for umbilical cable 228 to travel throughupper bearing assembly 130 of top case shell 126 down to cable guide 102(shown in FIG. 14). As discussed previously with respect to lowerbearing assembly 66, upper gear insert 131 may be any suitable geardesign for receiving an input rotation rate from a mating or contactinggear, such as the gears 110, 110′ of the mid-shaft gear assembly 108,with a size and tooth number selected to provide a desired gear train orspeed ratio when combined with contacting gears. For example, gearinsert 131 may be configured as a straight or spiral bevel gear, ahelical gear, a worm gear, a hypoid gear, and the like. In a preferredembodiment, gear 131 is a spiral gear to provide a smooth tooth actionat the operational speeds of the centrifugal processing system 10. Gearinsert 131 is preferably retained within column 137 by use of at leastone and preferably two spring pins (not shown); however, other assemblytechniques may be used to position and retain the gear insert 131 withinthe column 137 and such techniques are considered within the breadth ofthis disclosure. For example, gear insert 131 may be held in column 137by a number of other methods, such as, but not limited to being pressfit or frictionally fit or alternatively gear insert 131 and the upperbearing assembly may be molded from a unitary body.

Upper bearing assembly 130 is then inserted into axial bore 136 of topcase shell 126 so that the lower surface 135 sits flush with uppersurface 132 of top case shell 126. Ball bearing 128 is then insertedinto the annular space created between the outer diameter of column 137and the inner side wall 141 of top case shell 126 thereby securing upperbearing assembly 130 into place.

Referring now to FIG. 13, lower lip 134 is contoured to mate withprotrusions 88, 90, 92 and 94 extending from lower case shell 32.Specifically, the outer diameter of lower lip 134 matches the outerdiameter of the upper end of main body 40 of lower case shell 32 andrecesses 144 and 148 receive and retain protrusions 88 and 92respectively, while recesses 146 and 150 receive and retain protrusions94 and 88, respectively. Holes are placed through each recess and eachprotrusion so that when assembled, fasteners 152 (shown in FIG. 12) canbe inserted through the holes thereby fastening the top bearing assembly124 to the lower case assembly 30.

Positioned between recesses 144 and 146 and between recesses 148 and 150are recessed slots 104′ and 106′, respectively, for receiving gears 110and 110′ of mid-shaft gear assembly 108 (FIG. 2 and 14). The gears 110and 110′ are preferably configured to provide mating contact with thegear insert 131 and to produce a desired, overall gear train ratiowithin the centrifuge 20. In this regard, the gears 110 and 110′ arepreferably selected to have a similar configuration (e.g., size, toothnumber, and the like) as the gear 131, such as a spiral gear design.Furthermore recessed slots 96′ and 98′ exist between recesses 144 and150 and between recesses 146 and 148, respectively. When gears 110 and110′ are assembled as shown in FIG. 14, recessed slots 96 and 96′ fromthe lower case shell 32 and top case shell 126, respectively, form port154, and recessed slots 98 and 98′ form port 156 thereby allowing theumbilical cable 228 to exit centrifuge 20 through either port 154 or156. Described above is one method of assembling the centrifugalprocessing system 10 of the present invention; however, those skilled inthe art will appreciate that the lower case assembly 30 and upperbearing assembly can be joined in number of ways that allow the fourgears to be properly aligned with respect to one another.

In the above manner, the centrifugal processing system 10 provides acompact, portable device useful for separating blood and other fluids inan effective manner without binding or kinking fluid feed lines, cables,and the like entering and exiting the centrifuge 20. The compactness ofthe centrifugal processing system 10 is furthered by the use of theentirely contained and interior gear train described above thatcomprises, at least in part, gear 74, gears 110 and 110′, and gearinsert 131 of the upper bearing 130. The gear insert 131 of the upperbearing 130 is preferably selected to provide a contact surface(s) withthe gears 110 and 110′ that transfers the rotation rate of the gears 110and 110′ and consequently from gear 74 and to the gear insert 131 of theupper bearing 130. In one preferred embodiment, the gear insert 131 ofthe upper bearing 130 is a spiral gear rigidly mounted within the upperbearing 130 to rotate the rotor assembly 200 and having a design similarto that of the spiral gear 74, i.e., same or similar face advance,circular pitch, spiral angle, and the like. During operation, the gear74 remains stationary as the lower case shell 32 is rotated about thecentrifuge axis, A, at an input rotation rate, such as a rotation ratechosen from the range of 0 rpm to 5000 rpm. The gears 110, 110′ arerotated both about the centrifuge axis, A, with the shell 32 and bycontact with the stationary gear 74. The spiral gears 110, 110′ contactthe gear insert 131 of the upper bearing 130 causing the gear insert 131and connected upper bearing 130 to rotate at an output rotation ratethat differs, i.e., is higher, than the input rotation rate.

Although a number of gear ratios or train ratios (i.e., input rotationrate/output rotation rate) may be utilized to practice the invention,one embodiment of the invention provides for a gear train ratio of 1:2;where the combination and configuration of the gear 74, gears 110, 110′,and gear 131 of the upper bearing 130 are selected to achieve this geartrain ratio. Uniquely, the rotation of the gears 110, 110′ positivelyaffects the achieved gear train ratio to allow, in one embodiment, theuse of four similarly designed gears which lowers manufacturing costswhile achieving the increase from input to output rotation speeds.Similarly, as will be understood by those skilled in the mechanicalarts, numerous combinations of gears in differing number, size, andconfiguration that provides this ratio (or other selected ratios) may beutilized to practice the invention and such combinations are consideredpart of this disclosure. For example, although two gears 110, 110′ areshown in the mid-shaft gear assembly 108 to distribute transmissionforces and provide balance within the operating centrifuge, more (orless) gears may be used to transmit the rotation of gear 74 to the gearof the upper bearing 130. Also, just as the number, size, andconfiguration of the internal gears may be varied from the exemplaryillustration of FIGS. 1-14, the material used to fabricate the gear 74,the gears 110, 110′, and the gear insert 131 may be any suitable gearmaterial known in the art.

Another feature of the illustrated centrifugal processing system 10 thatadvantageously contributes to compactness is the side-mounted drivemotor 24. As illustrated in FIGS. 1 and 2, the drive motor 24 is mountedon the stationary base 12 of the enclosure 11 adjacent the centrifuge20. The drive motor 24 may be selected from a number of motors, such asa standard electric motor, useful for developing a desired rotation ratein the centrifuge 20 of the centrifugal processing system 10. The drivemotor 24 may be manually operated or, as in a preferred embodiment, amotor controller may be provided that can be automatically operated by acontroller of the centrifugal processing system 10 to govern operationof the drive motor 24 (as will be discussed in detail with reference tothe automated embodiment of the invention). As illustrated in FIG. 1, adrive belt 26 may be used to rotate the drive shaft assembly 28 (and,therefore, the rotor assembly 200). In this embodiment, the drive belt26 preferably has internal teeth (although teeth are not required toutilize a drive belt) selected to mate with the external teeth of thetiming belt ring 46 of the lower case assembly 30 portion of the driveshaft assembly 28. The invention is not limited to the use of a drivebelt 26, which may be replaced with a drive chain, an external geardriven by the motor 24, and any other suitable drive mechanisms. Whenoperated at a particular rotation rate, the drive motor 24 rotates thedrive shaft assembly 28 at nearly the same rotation rate (i.e., theinput rotation rate). A single speed drive motor 24 may be utilized orin some embodiments, a multi and/or variable speed motor 24 may beprovided to provide a range of input rotation rates that may be selectedby the operator or by a controller to obtain a desired output rotationrate (i.e., a rotation rate for the rotor assembly 200 and includedcentrifuge bag 226.

The present invention generally includes an apparatus and methods forthe separation of a predetermined fraction(s) from a fluid mediumutilizing the principles of centrifugation. Although the principles ofthe present invention may be utilized in a plurality of applications,one embodiment of this invention comprises isolating predeterminedfraction(s) (e.g., platelet rich plasma or platelet poor plasma) fromanticoagulated whole blood. The platelet rich plasma may be used, forexample, in the preparation of platelet concentrate or gel, and moreparticularly may be used to prepare autologous platelet gel duringsurgery using blood drawn from the patient before or during surgery.

The centrifuge 20 has been discussed above and demonstrates the compactand portable aspects of the present invention. To complete the device ofthe present invention a fluid collection device, also referred to as abowl or rotor 202 is attached to the upper surface 133 of the upperbearing assembly 130 as shown in FIGS. 1 and 2. Rotor 202 is preferablymounted permanently to upper bearing assembly 130, however, rotor 202may also be capable of being removed. Rotor 202 comprises a rotor base204 (shown in FIG. 15) having a lower annular groove 212, and a rotorcover 206 having an upper annular groove 214. As shown in FIGS. 17 and18 the annular interior chamber 216 of rotor 202 is defined by upper andlower annular grooves 212 and 214. The lower annular 212 receives acentrifuge bag 226 for containing the fluid medium to be centrifuged.Centrifuge bag 226 is connected to supply and receiving containers 398,400, respectively, via umbilical cable 228 which is preferably, but notlimited to a dual lumen. There may be instances where a certaintechnique requires multiple outlet or inlet ports and consequentlyumbilical cable 228 of the present invention may comprise multiplelumens. Umbilical cable 228 according to the preferred embodimentcomprises inlet lumen 230 and outlet lumen 232 such that a fluid mediummay be provided to and removed from the centrifuge bag 226 duringrotation of the centrifuge rotor 202.

One embodiment of centrifuge rotor 202 is more particularly illustratedin FIGS. 15, 16, 17 and 18. FIG. 15 is a perspective view of rotor base204, and FIG. 16 is a perspective view of rotor cover 206. FIG. 17 is across-sectional side view of rotor 202 taken along view lines 17 in FIG.1, and FIG. 18 is a cross-sectional side view of rotor 202 taken alongview lines 18 in FIG. 1. As illustrated in FIG. 15, rotor base 204comprises raised annular rim 208 and raised column 218 that is axiallydisposed in base 204. Raised column 218 further has a groove 222extending across the diameter of column 218. Annular groove 212 isdefined by raised annular rim 208 and raised column 218. The height ofrim 208 is equal to the height of column 218. Rotor cover 206 shown inFIG. 16 comprises raised annular rim 210 and raised column 220 which isaxially disposed in rotor cover 206. Raised column 220 further has agroove 224 extending across the diameter of column 220. Annular groove214 is defined by rim 210 and column 220. The height of rim 210 is equalto the height of column 220.

Generally, when centrifuge rotor 202 is to be assembled for use, aflexible centrifuge bag such as a doughnut-shaped centrifuge bag 226(FIG. 19 and 20) having a center core 242 is placed in rotor base 204such that center column 218 extends through the core 242 of centrifugebag 226 and the centrifuge bag 226 lies in annular groove 212. Rotorcover 206 is superimposed on rotor base 204 such that grooves 222 and224 are aligned, as illustrated in FIGS. 17 and 18. When rotor cover 204is secured to rotor base 206 by appropriate screws, fasteners, or thelike (not shown), rims 208 and 210 are in complete contact with eachother such that annular groove 212 and annular groove 214 define rotorinterior chamber 216. In one embodiment, columns 218 and 220 are incomplete contact with each other. Alternatively, the inner perimeter 240of centrifuge bag 226 is secured between columns 218 and 220 such thatcolumns 218 and 220 do not completely physically contact each other.

With the above description of one embodiment of the centrifuge in mind,another preferred embodiment of a centrifuge for use in the centrifugalprocessing system 10 will be described. Referring to FIGS. 19-22, apreferred embodiment of a centrifuge 640 is illustrated that utilizes auniquely arranged internal pulley system to obtain a desired input tooutput drive ratio (such as 2:1, as discussed above) rather than aninternal gear assembly. The centrifuge 640 utilizes the side-mountedmotor 24 (shown in FIG. 1) through drive belt 26 to obtain the desiredrotation rate at the rotor portion of the centrifuge.

Referring first to FIG. 19, the centrifuge 640 includes a rotor base 644(or top plate) with a recessed surface 648 for receiving and supportinga centrifuge bag during the operation of the centrifuge 640. The rotorbase 644 is rigidly mounted with fasteners (e.g., pins, screws, and thelike) to a separately rotable portion (i.e., a top pulley 698 discussedwith reference to FIGS. 20 and 21) of a lower case shell 660. A cableport 656 is provided centrally in the rotor base 644 to provide a pathfor a centrifuge tube or umbilical cable that is to be fluidicallyconnected to a centrifuge bag positioned on the recessed surface 648 ofthe rotor base 644. It is important during operation of the centrifuge640 to minimize and control contact and binding of the umbilical cableand moving parts (such as drive belts and pulleys). In this regard, thelower case shell 660 includes a side cable port 662 for the umbilicalcable to enter the centrifuge 640, which, significantly, the side cableport 662 is located between idler pulleys 666, 668 to provide a spacingbetween any inserted tube or cable and the moving drive components ofthe centrifuge 640.

Idler shaft or pins 664 are mounted and supported within the lower caseshell 660 to allow the pins 664 to physically support the pulleys 666,668. The idler pulleys 666, 668 are mounted on the pins 664 by bearingsto freely rotate about the central axis of the pins 664 during operationof the centrifuge 640. The idler pulleys 666, 668 are included tofacilitate translation of the drive or motive force provided or impartedby the drive belt 26 to the lower case shell 660 to the rotor base 644,as will be discussed in more detail with reference to FIGS. 20 and 21,and to physically support the internal drive belt 670 within thecentrifuge 640. The drive belt 26 is driven by the side-mounted motor 24(shown in FIG. 1) and contacts the lower case shell 660 to force thelower case shell 660 to rotate about its central axis. The lower caseshell 660 is in turn mounted on the base 674 in a manner that allows thelower case shell 660 to freely rotate on the base 674 as the drive belt26 is driven by the side-mounted motor 26. The base 674 is mounted to astationary base 12 (shown in FIG. 1) such that the base 674 issubstantially rigid and does not rotate with the lower case shell 660.

Referring now to FIGS. 20-22, the centrifuge 640 is shown with a cutawayview to more readily facilitate the discussion of the use of theinternal pulley assembly to obtain a desired output to input ratio, suchas two to one. As shown, the base 674 includes vibration isolators 676fabricated of a vibration absorbing material such as rubber, plastic,and the like through which the base 674 is mounted relatively rigidly tothe stationary base 12 (of FIG. 1). The drive belt 26 from theside-mounted motor 24 (of FIG. 1) contacts (frictionally or with the useof teeth and the like as previously discussed) a drive pulley 680, whichis rigidly mounted to the lower case shell 660. As the drive belt 26 isdriven by the motor 24, the lower case shell 660 through drive pulley680 rotates about its center axis (which corresponds to the center axisof the centrifuge 640). This rotation rate of the lower case shell 660can be thought of as the input rotation rate or speed.

To obtain a desired, higher rotation rate at the rotor base 644, thelower case shell 660 is mounted on the base to freely rotate about thecentrifuge center axis with bearings 690 that mate with the base 674.The bearings 690 are held in place between the bottom pulley 692 and thebase 674, and the bottom pulley 692 is rigidly attached (with bolts orthe like) to the base 674 to remain stationary while the lower caseshell 660 rotates. The illustrated bearings 690 are two piece bearingswhich allow the lower case shell 660 to rotate on the base 674. Aninternal drive belt 670 is provided and inserted through the lower caseshell 660 to contact the outer surfaces of the bottom pulley 692. Thebelt 670 preferably is installed with an adequate tension to tightlymate with the bottom pulley 692 such that frictional forces cause thebelt 670 to rotate around the stationary bottom pulley 692. Thisfrictional mating can be enhanced using standard rubber belts or beltswith teeth (and of course, other drive devices such as chains and thelike may be substituted for the belt 670).

The internal drive belt 670 passes temporarily outside the centrifuge640 to contact the outer surfaces of the idler pulleys 666 and 668.These pulleys 666, 668 do not impart further motion to the belt 670 butrotate freely on pins 664. The idler pulleys 666, 668 are included toallow the rotation about the centrifuge center axis by lower case shell660 to be translated to another pulley (i.e., top pulley 698) thatrotates about the same axis. To this end, the idler pulleys 666, 668provide non-rigid (or rotable) support that assists in allowing the belt670 to be twisted without binding and then fed back into an upperportion of the lower case assembly 660 (as shown clearly in FIGS. 20 and21). As the internal drive belt 670 is fed into the lower case assembly660, the belt 670 contacts the outer surfaces of a top pulley 698.

During operation of the centrifuge 640, the movement of the internaldrive belt 670 causes the top pulley 698 to rotate about the centrifugecenter axis. The idler pulleys 666 and 668 by the nature of theirplacement and orientation within the centrifuge 640 relative to thepulleys 692 and 698 cause the rotor base 644 to rotate in the samedirection as the lower case shell 660. Significantly, the top pulley 698rotated about the centrifuge center axis at twice the input rotationrate because it is mounted to the lower case shell 660 via bearings 694(preferably, a two piece bearing similar to bearings 690 but otherbearing configurations can be used) which are mounted to the centershaft 686 of the lower case shell 660 to frictionally contact an innersurface of the top pulley 698. Since the internal drive belt 670 isrotating about the bottom pulley 692 and the idler pulleys 666, 668 arerotating about the centrifuge central axis by drive belt 26, the toppulley 698 is turned about the centrifuge central axis in the samedirection as the lower case shell 660 but at twice the rate.

In other words, the drive force of the drive belt 26 and the internaldrive belt 670 are combined by the components of the centrifuge 640 tocreate the output rotation rate. While a number of output to input driveratios may be utilized, as discussed previously, a 2:1 ratio isgenerally preferable, and the centrifuge 640 is preferably configuredsuch that the second, faster rotation rate of the top pulley 698 issubstantially twice that of the lower case shell 660. The use of aninternal drive belt 670 in combination with two pulleys rotating aboutthe same axis and the structural support for the pulleys within arotating housing results in a centrifuge that is very compact and thatoperates effectively at a 2:1 drive ratio with relatively low noiselevels (which is desirable in many medical settings).

The 2:1 drive ratio obtained in the top pulley 698 is in turn passed onto the rotor base 644 by rigidly attaching the rotor base 644 to the toppulley 698 with fasteners 652. Hence, a centrifuge bag placed on therecessed surface 648 of the rotor base 644 is rotated at a rate twicethat of the umbilical cable 228 that is fed into lower case shell 660,which effectively controls binding as discussed above. The bearing 694(one or more pieces) wrap around the entire center shaft 686 of thelower case shell 660. To provide a path for the umbilical cord 228 topass through the centrifuge 640 to the rotor base 644 (which duringoperation will be enclosed with a rotor top or cover as shown in FIG.1), the rotor base 644 includes the cable port 656 and the center shaft686 is configured to be hollow to form a center cable guide. This allowsan umbilical cable 228 to be fed basically parallel to the centrifugecenter axis to the centrifuge bag (not shown). The lower case shell 660includes the side cable port 662 to provide for initial access to thecentrifuge 640 and also includes the side cable guide (or tunnel) 684 toguide the cable 228 through the lower case shell 660 to the hollowportion of the center shaft 686. The side port 662 and the side cableguide 684 are positioned substantially centrally between the two idlerpulleys 666, 668 to position the cable 228 a distance away from theinternal drive belt 670 to minimize potential binding and wear.

The centrifuge 640 illustrated in FIGS. 19-22 utilizes two piecebearings for both the bottom and top pulleys 692 and 698, respectively,and to provide a path for the umbilical cable 228 a central “blind”pathway (via side cable guide 684, the hollow center of the center shaft686, and cable ports 656, 662) was provided in the centrifuge 640. Whileeffective, this “blind” pathway can in practice present binding problemsas the relatively stiff cable 228 is fed or pushed through the pathway.To address this issue, an alternate centrifuge embodiment 700 isprovided and illustrated in FIGS. 23 and 24. In this embodiment, theupper portions of the centrifuge 700 include a guide slot between theidler pulleys 666, 668 that enables an umbilical cable 228 to be fedinto the centrifuge 700 from the top with the no components to block theview of the operator inserting the cable 228.

To allow a guide slot to be provided, the contiguous upper bearing 694in the centrifuge 640 are replaced with bearing members that have atleast one gap or separation that is at least slightly larger than theouter diameter of the cable 228. A number of bearing members may beutilized to provide this cable entry gap and are included in the breadthof this disclosure. As illustrated, the centrifuge 700 includes a rotorbase 702 that is rigidly fastened with fasteners 704 to the top pulley698 (not shown) to rotate with this pulley at the output rate (e.g.,twice the input rate) and to receive and support a centrifuge bag onrecessed surface 716. The rotor base 702 further includes the cable port718 which is useful for aligning the center of the bag and cable 228with the center of the centrifuge 700.

To allow ready insertion of the cable 228 in the centrifuge 700, therotor base 702 further includes a cable guide slot 712 which asillustrated is a groove or opening in the rotor base 702 that allows thecable 228 to be inserted downward through the centrifuge 700 toward theside cable guide 724 of the lower case shell 720. The lower case shell720 also includes a cable guide slot 722 cut through to the top of theside cable guide 724. Again, the guide slots 712 and 724 are bothlocated in a portion of the centrifuge 700 that is between the idlerpulleys 666, 668 to position an inserted cable 228 from contacting andbinding with the internal drive belt 670, which basically wraps around180 degrees of the top pulley or lower case shell 720.

As shown in FIG. 23, the bearing members 706 are spaced apart andpreferably, at least one of these spaces or gaps is large enough to passthrough the cable 228 to the center shaft of the lower case shell 720.As illustrated, four cam followers are utilized for the bearing members706, although a different number may be employed. The cam followers 706are connected to the top pulley to enable the top pulley to rotate andare connected, also, to the center shaft of the lower case shell 720 torotate with the lower case shell 720. The cam followers 706 ride in abearing groove 710 cut in the lower case shell 720. To provide anunobstructed path for the cable 228, the cable guide slots 712 and 722are positioned between the two cam followers 706 adjacent the idlerpulleys 666, 668, and preferably the guide slots 712, 722 are positionedsubstantially centrally between the pulleys 666, 668. The guide slots712, 722 are positioned between these cam followers 706 to position thecable 228 on the opposite side of the centrifuge 700 as the contactsurfaces between the internal drive belt 670 and the top pulley 698(shown in FIGS. 20-22). In this manner, the use of separated bearingmembers 706 in combination with a pair of cable guide slots 712, 722allows an operator to readily install the umbilical cable 228 withouthaving to blindly go through the inside of the drive system andminimizes binding or other insertion difficulties.

A. Flexible, Disposable Centrifuge Bag

One embodiment of disposable, flexible centrifuge bag 226 is moreparticularly illustrated in FIGS. 25 and 26. The bag is an integral twostage self balancing disposable design. The disposable centrifuge bag226 has a substantially flat, toroidal- or doughnut-shaped configurationhaving outer and inner perimeters 238 and 240, respectively, andcomprises radially extending upper and lower sheets 234, 236 formed froma substantially flexible material. The upper and lower sheets 234, 236are superimposed and completely sealed together at outer perimeter 238by a heat weld, rf (radio frequency) weld or other comparable method ofadhering two surfaces. Inner perimeter 240 defines core 242 of bag 226.In one embodiment of the invention, centrifuge bag 226 further comprisesan inlet tube 248 sandwiched between upper and lower sheets 234, 236 andextending from the center of core 242 defined by inner perimeter 240 tothe outer perimeter 238 and an outlet tube 250 sandwiched between upperand lower sheets 234, 236 and extending from the center of the core 242to the outer perimeter 238. When upper and lower sheets 234, 236 aresealed together at inner perimeter 240, inlet and outlet tubes 248, 250are thereby sealed therebetween. Inlet and outlet tubes 248, 250 areeach in fluid communication with the interior of centrifuge bag 226 andthe environment outside centrifuge bag 226. The length of outlet tube250 is shorter than the length of inlet tube 248.

In one embodiment of this invention, outlet tube 250 is a straight tubeas shown in FIG. 31. Alternatively, outlet tube 250 includes a bentfitting 252 fluidly connected to the distal end of outlet tube 250(FIGS. 25 and 26). The bent fitting 252 may be of any number ofconfigurations, although preferably bent fitting 252 is shaped in theform of a “T”, “curved T”, a “J”, or an “L”, as illustrated in FIGS. 27,28, 29 and 30, respectively. Alternatively, outlet tube 250 and bentfitting 252 may be one contiguous molded unit rather than two connectedpieces. Preferably, bent fitting 252 is in the shape of a “T” or a“curved T” as illustrated in FIGS. 27 and 28, respectively. The “T” or“curved T” design of bent fitting 252 ensures that the desired bloodcomponent (fraction) will be removed from the sides of the bent fitting252, rather than from a fraction located above or below the bentfitting, as discussed below in detail.

When the centrifuge bag 226 is positioned in the annular groove 212 ofthe centrifuge rotor 202 as described above, it is critical that inletand outlet tubes 248, 250 are seated in groove 222. Further, when rotorcover 206 is positioned over and removably secured to the centrifugebase 204, it is important that inlet and outlet tubes 248, 250 are alsoseated in groove 224. Seating inlet and outlet tubes 248, 250 in grooves222, 224 ensures that centrifuge rotor 202 is held in a fixed positionbetween rotor base 204 and rotor cover 206 such that the centrifuge bag226 and centrifuge rotor 202 rotate together. That is, the fixedposition of centrifuge bag 226 ensures that centrifuge bag 226 will notrotate independently of centrifuge bag 226 during centrifugation.

Inlet and outlet tubes 248, 250 are fluidly connected at their proximalends to umbilical cable 228, which in this particular embodiment is adual lumen tubing connecting centrifuge bag 226 to source and receivingcontainers 398, 400, respectively, for the introduction and removal ofcomponents from the centrifuge bag 226 during centrifugation (see FIG.17). Dual lumen tubing 228 comprises inlet lumen 230, which connectsinlet tube 248 of centrifuge bag 226 with source container 398, andoutlet lumen 232, which connects outlet tube 250 centrifuge bag 226 withreceiving container 400. In one embodiment, the inlet and outlet tubes248, 250 are adapted at their proximal ends for inserting into the inletand outlet lumens 230 and 232, respectively. Alternatively, connectingmeans 254 are inserted into the proximal ends of inlet and outlet tubes248, 250 for connecting the tubes to the inlet and outlet lumens 230,232 as illustrated in FIG. 26.

In operation, one end of umbilical cable 228 must be secured to rotorassembly 200 to prevent itself from becoming twisted during rotation ofrotor assembly 200 by the coaxial half-speed rotation of drive shaftassembly 28, which imparts a like rotation with respect to the rotor 202axis and consequently to the umbilical cable 228 that is directedthrough cable guide 102. That is, if rotor assembly 200 is considered ashaving completed a first rotation of 360° and drive shaft assembly 28 ashaving completed a 180° half-rotation in the same direction, theumbilical cable 228 will be subjected to a 180° twist in one directionabout its axis. Continued rotation of rotor assembly 200 in the samedirection for an additional 360° and drive shaft assembly 28 for anadditional 180° in the same direction will result in umbilical cable 228being twisted 180° in the opposite direction, returning umbilical cable228 to its original untwisted condition. Thus, umbilical cable 228 issubjected to a continuous flexture or bending during operation of thecentrifugal processing system 10 of the present invention but is nevercompletely rotated or twisted about its own axis.

An alternative embodiment of a disposable centrifuge bag of thisinvention, shown in FIG. 35 comprises two or more inlet tubes and/or twoor more outlet tubes, wherein the tubes are fluidly connected to amultiple lumen tubing.

The disposable centrifuge bag 226 is formed from a transparent,substantially flexible material, including but not limited to, polyvinylchloride, polyethylene, polyurethane, ethylene vinyl acetate andcombinations of the above or other flexible materials. Based upon theflexibility of the centrifuge bag 226, the profile of the flexiblecentrifuge bag 226, shown in FIGS. 25 and 26, is determined at least inpart by the amount of fluid contained therein. The profile of centrifugebag 226 is further defined by the interior configuration of thecentrifuge rotor, as discussed below in detail. The ability tomanipulate the profile of centrifuge bag 226 based on the interiorconfiguration of the centrifuge rotor is utilized at least in part tomaximize the volume of fluid medium that can be contained in centrifugebag 226 during centrifugation, as will be discussed below.

The fluid or medium to be centrifuged may be contained within sourcecontainer 300. For example, when the centrifuge 20 of this invention isused to prepare an autologous platelet gel, the fluid (i.e., wholeblood), may be withdrawn from the patient during or prior to surgeryinto source container 398 containing an anticoagulant. Theanticoagulated whole blood is introduced to centrifuge bag 226 throughinlet tube 248 via inlet lumen 230 after the centrifuge bag 226 has beenpositioned in the centrifuge rotor 202 and rotation thereof isinitiated. As discussed above, securing centrifuge bag 226 in centrifugebase 204 in grooves 222, 224 holds the centrifuge bag 226 in a fixedposition therebetween, such that the centrifuge bag 226 cannot moveindependently of the centrifuge rotor 202, and therefore the centrifugebag 226 and rotor assembly 200 rotate concurrently at the same rate ofrotation. Rotation of the centrifuge rotor 202 directs the heavierdensity constituents of the anticoagulated whole blood within thecentrifuge bag 226 toward the outer perimeter 238 of the bag 226, whilethe lighter density constituents remain closer to an inner region, asillustrated in FIG. 32. More specifically, as illustrated in FIG. 32,when the fluid medium being separated is whole blood, the whole blood isseparated within centrifuge bag 226 into a red blood cell fraction(256), a white blood cell fraction (258), a platelet rich plasmafraction (260), and a platelet poor plasma fraction (262). As will beappreciated by those of skill in the art, whole blood fractions, redblood cell's and plasma are differently colored, and consequently theseparation of the fractions can be easily detected by the operator. Atan appropriate time during centrifuging, suction or other drawing meansmay be applied to the interior of centrifuge bag 226 via outlet lumen232 to remove the desired fraction from the centrifuge bag 226. In afurther embodiment, centrifuge cover 206 may further contain concentricindex lines to assist the operator in viewing the positions of outlettube 250 to the RBC plasma interface. Based on the speeds and times thelocation of the WBC and platelets can be varied with respect to the redblood cell's and plasma interface. For example, if the rpm is held low(approximately 1,000-1,700, preferably 1,500) the plasma and plateletswill separate from the RBC layer, as the rpm's are increased(1,400-1,700) the platelets will separate out of the plasma and resideat the plasma to RBC interface in greater concentrations. With increasedspeeds WBC reside deeper into the RBC pack.

With further regard to bent fittings 252, in one embodiment a bentfitting is fluidly connected to the distal end of outlet tube 250. Whilebent fitting 252 is shown in FIG. 32 as having a “T” shape (FIG. 27),this is for illustrative purposes only. Thus, it will be appreciatedthat bent fitting 252 as shown in FIG. 32 could have a number of otherconfigurations, such as those shown in FIGS. 25-31. The design of bentfitting 252 ensures that the desired component is withdrawn (e.g., theplatelet rich plasma fraction 260) with less risk of contamination fromwithdrawing a portion of the adjacent fraction 258. Thus, in oneembodiment, the desired fraction is withdrawn when its position overlapswith the position of bent fitting 252. Alternatively, the inlet tube 248may be first used to draw off the red blood cell fraction 256, and whenit is desirable to remove the predetermined fraction from the centrifugebag 226, the predetermined fraction is drawn through bent fitting 252and outlet tube 250 and directed to receiving container 400 via outletlumen 232.

With continued reference to FIG. 32, as the separation of the fluidmedium is initiated by centrifugation, substantially annular regionshaving constituents of a particular density or range of densities beginto form. For purposes of illustration, the separation of whole bloodwill be discussed, and as shown in FIG. 32 four regions are represented,each of which contains a particular type of constituent of a givendensity or range of densities. Moreover, it should be appreciated thatthere may be a given distribution of densities across each of theregions such that the regions may not be sharply defined. Consequently,in practice the regions may be wider (e.g., a larger radial extent) andencompass a range of densities of constituents.

In the example of FIG. 32, the first region 256 is the outermost of thefour regions and contains red blood cells. The second region 258contains white blood cells, which have a lower density than that of thered blood cells. The third region 260 contains the platelet rich plasmafraction, and the innermost region 262 contains the least dense plateletpoor plasma fraction. In one embodiment, it may be desired to harvestthe platelet rich plasma fraction in region 260. In order to remove theplatelet rich plasma fraction from the centrifuge bag 226, vacuum orsuction is provided via outlet lumen 232 to the centrifuge bag 226 toremove a desired portion of region 260. A portion of the fraction 260that is in the area of the bent fitting 252 is drawn through bentfitting 252 and into an appropriate one of the collection containers 400(FIG. 17).

More specifically, FIGS. 33-39 illustrate one method of this inventionfor the separation of whole blood components, which is a dynamicprocess. FIG. 33 shows one portion of the centrifuge bag 226,illustrating the separation of the whole blood components after infusionof an aliquot of whole blood into centrifuge bag 226 and centrifugationfor approximately 60 seconds to 10 minutes at a rate of rotation between0 and 5,000 rpms. It will be understood by those of skill in the artthat faster speeds of rotation will separate the blood in a shorterprior of time.

FIG. 33 shows the four separated whole blood fractions, with the denserfractions closer to outer perimeter 238, and the less dense fractionscloser to inner perimeter 240. While it is well-known that hematocrits(i.e., the volume of blood, expressed as a percentage, that consists ofred blood cells) will vary among individuals, ranging from approximately29%-68%, such variations are easily adjusted for as a result of thenovel design of centrifuge bag 226 and consequently will not affect theisolation of any of the desired fractions as discussed below in detail.Thus, for illustrative purposes, it will be assumed that centrifugationof an initial infusion of an aliquot of anticoagulated whole blood willgive the profile shown in FIG. 33. In one embodiment, it is desired toharvest the platelet rich plasma fraction 260. This may be achieved byperforming a batch separation process or a continuous separation processas described below.

In one embodiment of a batch separation process of this invention forharvesting the platelet rich plasma fraction 260, centrifuge bag 226 hasa design as shown in FIG. 32 wherein bent fitting 252 positionedapproximately in the area where a platelet rich plasma fraction 260 istypically found after centrifugation of an aliquot of whole blood. Thisapproximation is simplified by the placement of concentric indicatorlines 205, 207, and 209, (not shown) in the upper surface of rotor cover206, wherein the concentric lines 205, 207 and 209 correspondapproximately with the edges of regions 260, 258, and 256, respectively.Alternative, concentric lines similar to 205, 207 and 209 may bedirectly imprinted onto the surface of centrifuge bag 226.

After centrifugation of an aliquot of blood contained in centrifuge bag226, a substantial portion of the platelet rich plasma fraction 260 iswithdrawn from centrifuge bag 226 through bent fitting 252 whilecentrifuge rotor 202 is still spinning. As the volume of the plateletrich plasma fraction 260 is reduced upon withdrawal, the innermostfraction 262 naturally moves in the direction of the outer perimeter 238due to centrifugal force, as shown in FIG. 34. The withdrawal ofplatelet rich plasma fraction 260 is terminated at a point where theplatelet poor plasma fraction 262 is close to bent fitting 252 andbefore any significant portion of platelet poor plasma fraction 262could be withdrawn through bent fitting 252, as shown in FIG. 34. Thispoint can be determined either visually by the operator by volume, or bya sensor, as described below in detail. After withdrawal of the desiredplatelet rich plasma fraction 260, inlet lumen 230 is disconnected fromthe whole blood source container 398 and connected to a disposalcontainer, after which the remaining fluid in centrifuge bag 226 isevacuated through inlet tube 248 and directed to the disposal container.The inlet lumen is then reconnected to the whole blood source container,and the above-described batch process is repeated as many times asrequired until the necessary quantity of the desired fraction isisolated.

Alternatively, the above-described process can be performed as acontinuous process wherein the step of disconnecting the inlet lumen 230from the whole blood source 398 can be avoided. The continuous processseparation of whole blood may be achieve by using a disposablecentrifuge bag 226′ as illustrated in FIGS. 39-39 comprising an inlettube 248 and three outlet tubes 245, 247 and 250, wherein the tubes areconnected to an umbilical cable comprising four lumens. Morespecifically, a disposable centrifuge bag for use in a continuousseparation of whole blood comprises inlet tube 248 connected via aninlet lumen to a whole blood source container, a first outlet tube 250connected to a first outlet lumen that is in turn connected to aplatelet rich plasma receiving container, a second outlet tube 245connected via a second outlet lumen to either a red blood cell receivingcontainer or a waste container and a third outlet tube 247 connected viaa third outlet lumen to a platelet poor plasma receiving container. Inthe continuous separation process, after withdrawal of the portion ofplatelet rich plasma or other cellular components as described abovewith reference to FIGS. 33 and 34. Centrifuge bag has the capacity toreceive an additional volume (aliquot) of whole blood. Consequently, asshown in FIG. 35 infusion of an aliquot of whole blood is reinitiatedthrough first inlet tube 248 with continued centrifugation until thecapacity of the centrifuge bag 226′ is reached. As a result of theadditional volume of blood, the profile of the blood fractions incentrifuge bag 226′ will approximately assume the profile shown in FIG.35. As can be seen in FIG. 35, the additional volume of blood results ina shift of the location of the blood fractions, such that the plateletrich plasma fraction 260 has shifted back into the area of the bentfitting 252, and the platelet poor plasma fraction 262 has shifted backtowards the inner perimeter 240 and away from the vicinity of the bentfitting 252. Additional platelet rich plasma 260 can now be removed fromcentrifuge bag 226′ through outlet tube 250 as shown in FIG. 35.

As described above, removal of an additional volume of the platelet richplasma fraction 260 results in a shift in the location of the plateletpoor plasma fraction 262 closer to the outer perimeter 238 andconsequently closer to the vicinity of bent fitting 252, as shown inFIG. 36, at which point removal of platelet rich plasma is againtemporarily terminated.

Additional infusions of whole blood aliquots to centrifuge bag 262′ andremoval of platelet rich plasma (by shifting the position of theplatelet rich plasma fraction 260 relative to the position of the bentfitting 252) as described above may be repeated a number of times.Eventually, however, the continued infusion of whole blood followed byremoval of only the platelet rich plasma fraction will necessarilyresult in a gradual increase in the volumes (and consequently thewidths) of the remaining blood fractions 256, 258 and 260 in centrifugebag 226′. In particular, the volume, and therefore the width, of the redblood cell fraction 256 will increase to the extent that the otherfractions are pushed closer to the inner perimeter 240 (FIG. 37). Asshown in FIG. 37, the increased volume of red blood cells now present incentrifuge bag 226′ shifts the location of the fractions towards theinner perimeter 240 such that the white blood cell fraction 260 is nowin the vicinity of the bent fitting 252 as opposed to the desiredplatelet rich plasma fraction 262.

The novel design of centrifuge bag 226′ advantageously provides meansfor shifting the fractions back to the desired locations when thesituation shown in FIG. 37 arises. That is, second outlet tube 245serves as an inlet conduit for introduction of whole blood aliquots intocentrifuge bag 226′, also serves the function of withdrawing fractionsthat are located close to the outer perimeter 238. This is achieved inpart by attaching the second outlet lumen to either a red blood cellreceiving container or a waste container having a suction means (e.g.,syringe, pump, etc.). As shown in FIG. 38, second outlet tube 245,having its distal end close to outer perimeter 238, can be operated towithdraw a substantial volume of the red blood cell fraction 256, whichin turn shifts the location of the remaining fractions 258, 260, 262.The withdrawal of the red blood cell fraction 256 may be monitoredvisually by the operator, or by other means such as a sensor.Alternatively, the positions of the fractions may be shifted bywithdrawing the platelet poor plasma fraction 262 through third outlettube 247, which is connected via a third outlet lumen to a platelet poorplasma receiving container.

FIG. 37 shows that, after withdrawal of a portion of the red blood cellfraction 256, the centrifuge bag 226′ again has the capacity to receivean additional volume of whole blood for centrifugation. An additionalinfusion of an aliquot of whole blood through inlet tube 248 into thecentrifuge bag 226′ of FIG. 37 and centrifugation will produce theprofile illustrated in FIG. 39. The above-described steps may berepeated as needed until the desired amount of platelet rich plasma hasbeen harvested. All of the above-described steps occur while thecentrifuge rotor 202 is spinning.

The above-described continuous separation method was illustrated interms of performing the whole blood infusion step and the platelet richplasma harvesting step sequentially. An alternative embodiment involvesperforming the infusion and harvesting steps substantiallysimultaneously, that is, the platelet rich plasma fraction is withdrawnat approximately the same time as an additional aliquot of whole bloodis being added to the bag. This alternate embodiment requires that thecentrifuge rotor spin at a rate that results in almost immediateseparation of the blood components upon infusion of an aliquot of wholeblood.

As stated previously, all of the above-described steps may be monitoredeither visually by the operator by volume, or by a sensor. If the stepsare to be visually monitored, centrifuge cover 206 may further includeone or more concentric indicator circles 205, 207, 209 (shown in FIGS.17 and 18) which may be spaced from the center of cover 206 at distancesapproximately equal to the outer edges of regions 260, 258 256,respectively, to aid the operator in visualizing the positions of theseregions with respect to 252.

FIGS. 33-39 illustrate one embodiment of how the design of centrifugebags 226 and 226′ permit the general locations of the various bloodfractions to be shifted to allow for continuous harvesting of a desiredblood fraction without the risk of contaminating the harvested bloodfraction, and further allow for continual on-line harvesting of a largevolume (10 to 5 L's) of blood using a small, portable centrifuge devicecomprising a 10 cc to 200 cc capacity disposable centrifuge bags 226 and226′.

For example, the design of centrifuge bag 226 having inlet tube 248 andoutlet tube 250 means that the desired component or fraction will bewithdrawn from centrifuge bag 226 only through outlet tube 250, whilethe addition of whole blood aliquots or the removal of other components(e.g., red blood cell fraction 256) will proceed only through dualfunctional inlet tube 248. In this respect, the harvested fraction(e.g., platelet rich plasma fraction 260) is never withdrawn throughinlet tube 248 which was previously exposed to other fluid media (e.g.,whole blood or red blood cells). Thus, the design of centrifuge bag 226offers a significant advantage over conventional centrifuge containerscomprising only one tube which serves to both introduce the fluid mediumto the container and to withdraw the harvested fraction from thecontainer.

Furthermore, because of its unique design, the use of centrifuge bags226 and 226′ are independent of composition of the whole blood to becentrifuged. For example, as stated above, hematocrits (i.e., thepercent volume of blood occupied by red blood cells) vary fromindividual to individual, and consequently the profile illustrated inFIG. 32 will vary from individual to individual. That is, the width ofred blood cell fraction 256 may be wider or narrower, which in turn willresult in the platelet rich plasma fraction 260 being positioned furtheraway in either direction from bent fitting 252. However, as discussedabove in detail with particular reference to FIGS. 33-34, the design ofcentrifuge bags 226 and 226′ allow the location of the desired fractionto be shifted until it is in the region of bent fitting 252. Suchshifting can be brought about, for example using centrifuge bag 226, bywithdrawing the red blood cell fraction through inlet tube 248, or byadding whole blood aliquots through inlet tube 248.

An alternative embodiment of a disposable, flexible centrifuge bag 270is illustrated in FIG. 40. The disposable centrifuge bag 270 has asubstantially flat, toroidal- or doughnut-shaped configuration havingouter and inner perimeters 271 and 272, respectively, and comprisesradially extending upper and lower sheets 273, 274 formed from asubstantially flexible material. The upper and lower sheets 273, 274 aresuperimposed and completely sealed together at outer perimeter 271 by anrf weld, heat weld or other comparable method of adhering two surfaces.Inner perimeter 272 defines core 275 of centrifuge bag 270. In oneembodiment of the invention, centrifuge bag 270 further comprises inlettube 276 sandwiched between upper and lower sheets 273, 274 and radiallyextending from the center of core 275 to the outer perimeter 271, andoutlet tube 278 sandwiched between upper and lower sheets 273, 274 andextending across the diameter of core 275 and having first and seconddistal ends 280, 281. When upper and lower sheets 273, 274 are sealedtogether at inner perimeter 272, inlet and outlet tubes 276, 278 arethereby sealed therebetween. Inlet and outlet tubes 276, 278 are each influid communication with the interior of centrifuge bag 270 and theenvironment outside centrifuge bag 270. Inlet tube 276 and outlet tube278 are fluidly connected to umbilical cable 228 (not shown), which inthis particular embodiment is a dual lumen tubing. Inlet tube 276 isfluidly connected at its proximal end to umbilical cable 228, preferablyby an L-shaped connector (not shown), and outlet tube 278 is fluidlyconnected at its center to umbilical cable 228 via a T-shaped connector(not shown).

The disposable centrifuge bag 270 is formed from a transparent,substantially flexible material, including but not limited to, polyvinylchloride, polyethylene, polyurethane, ethylene vinyl acetate andcombinations of the above or other flexible materials.

Upper and lower sheets 273, 274 of centrifuge bag 270 are further sealedat two portions between the outer perimeter and the inner perimeter.That is, centrifuge bag 270 further comprises a first C-shaped seal 282located between the outer and inner perimeters 271, 272 and having anfirst concave indentation or well 283 on the concave side of C-shapedseal 282, and a second C-shaped seal 284 located between the outer andinner perimeters 271, 272 and having an second concave indentation orwell 285 on the concave side of C-shaped seal 284. First and secondC-shaped seals 282 and 284 are formed by sealing portions of upper andlower sheets 273, 274 together by methods known in the art for sealingtwo surfaces, including but not limited to rf or heat welding. Ends 288and 289 of first C-shaped seal 282 are bent inward towards the innercore 275, and likewise ends 290 and 291 of second C-shaped seal 284 arebent inward towards the inner core 275. First and second C-shaped seals282, 284 have their concave sides facing each other such that the firstand second indentations 283, 285 are diametrically opposed to eachother. That is, when centrifuge bag 270 is viewed from the top as inFIG. 40, first and second C-shaped seals 282, 284 are mirror images ofeach other. First and second C-shaped seals 282, 284 together define anouter chamber 292 between the outer perimeter 271 and first and secondC-shaped seals 282, 284, wherein the outer chamber 292 has a toroidalconfiguration and serves as a first processing compartment. First andsecond C-shaped seals 282, 284 together further define an inner chamber293 between first and second C-shaped seals 282, 284 and inner perimeter272, wherein the inner chamber 293 has a toroidal configuration andserves as a second processing compartment. The first and second C-shapedseals 282, 284 are positioned such that ends 288 and 290 are directlyopposite and spaced apart from each other to define a first channel 286therebetween, and such that ends 289 and 291 are directly opposite andspaced apart from each other to define a second channel 287therebetween, wherein the first and second channels 286, 287 arediametrically opposed and provide fluid communication between the firstprocessing compartment 292 and the second processing compartment 293.Inlet tube 276 extends through either channel 286 or channel 287, andthe first and second distal ends 280, 281 of outlet tube 278 extend intofirst and second indentations 283, 285, respectively.

Centrifuge bag 270 is removably secured between rotor base 204 and rotorcover 206 of rotor 202 in a manner as described above so that centrifugebag 270 is held in a fixed position relative to rotor base 204 and rotorcover 206 during rotation of the centrifuge rotor 202. As will beappreciated by those of skill in the art, alternative embodiments ofrotor base 204 (FIG. 15) and rotor cover 206 (FIG. 16) will be requiredto accommodate the design of centrifuge bag 270. Thus, an alternateembodiment of rotor base 204 comprises raised column 218 comprisingfirst and second grooves which are perpendicular to each other andextend the diameter of the raised base column 218, such that when rotor202 is assembled, inlet tube 276 and outlet tube 278 of centrifuge bag270 are seated in the first and second grooves, respectively, of raisedbase column 218. Similarly, an alternate embodiment of cover 206comprises raised column 220 comprising first and second grooves whichare perpendicular to each other and extend the diameter of the raisedcover column 220, such that when rotor 202 is assembled, inlet tube 276and outlet tube 278 are further seated in the first and second grooves,respectively, of raised cover column 220.

As stated above, inlet and outlet tubes 276, 278 are fluidly connectedto umbilical cable 228, which in this particular embodiment is a duallumen tubing connecting centrifuge bag 270 to source and receivingcontainers 398, 400, respectively, for the introduction of the fluid tobe centrifuged in bag 270 and for the removal of one or more of theseparated components from the centrifuge bag 270 during rotation of thecentrifuge 20. Dual lumen tubing 228 comprises inlet lumen 230, whichconnects inlet tube 276 with source container 398, and outlet lumen 232,which connects outlet tube 278 with receiving container 400.

The fluid or medium to be centrifuged using centrifuge bag 270 may becontained within source container 398. For example, when the centrifuge20 of this invention is used to prepare an autologous platelet gel, thefluid (i.e., whole blood), may be withdrawn from the patient during orprior to surgery into source container 398 containing an anticoagulant.The anticoagulated whole blood is introduced to centrifuge bag 270through inlet tube 276 via inlet lumen 230 after the centrifuge bag 270has been positioned in the centrifuge rotor 202 and rotation thereof isinitiated.

Centrifuge bag 270 may be used for the separation and isolation of oneor more components dissolved or suspended in a variety of fluid media,including, but not limited to, the separation of cellular componentsfrom biological fluids. For example, centrifuge bag 270 is useful forthe concentration and removal of platelets from whole blood. Therefore,the following description of the separation of platelets from wholeblood using centrifuge bag 270 is merely for purposes of illustrationand is not meant to be limiting of the use of bag 270. The separation ofa fluid medium such as whole blood in centrifuge bag 270 may beconsidered to be a two-stage separation process. The first stage of theseparation of platelets from whole blood involves separation of aplatelet suspension from the red blood cells. The platelet suspension istypically plasma rich in platelets, and it is commonly referred to asplatelet-rich plasma (PRP). However, as used herein, the term “plateletsuspension” is not limited to PRP in the technical sense, but isintended to encompass any suspension in which platelets are present inconcentrations greater than that in whole blood, and can includesuspensions that carry other blood components in addition to platelets.The second stage of the separation comprises separating platelets fromthe platelet suspension to produce a platelet concentrate. As usedherein, the term “platelet concentrate” is intended to encompass avolume of platelets that results after a “platelet suspension” undergoesa subsequent separation step that reduces the fluid volume of theplatelet suspension. The platelet concentrate may be a concentrate thatis depleted of white blood cells and red blood cells.

With reference to FIG. 41, stage one of a whole blood separation processusing centrifuge bag 270 begins with the introduction of an aliquot ofwhole blood into centrifuge bag 270 via inlet tube 276 during rotationof the centrifuge 20. As the aliquot of whole blood enters outer chamber292 of centrifuge bag 270, it quickly separates radially under theinfluence of centrifugal force into various fractions within outerchamber 292 based on the densities of the components of the whole blood,including an outermost fraction containing the red blood cells whichpack along the outer perimeter 271 of centrifuge bag 270, and an innerfraction comprising the platelet suspension. The platelet suspensionafter centrifugation of the first aliquot of whole blood is representedin FIG. 41 by ring 294. Continued infusion of whole blood into the firstprocessing compartment 292 adds an additional volume of red blood cellsand consequently pushes the platelet suspension inward as represented byring 295. Additional infusions of whole blood will continue to push theplatelet suspension further inward, as represented by rings 296 and 297until the first processing compartment 292 is substantially filled withred blood cells (the remainder of the volume being plasma) such that theplatelet suspension is pushed through channels 286 and 287 into secondprocessing compartment 293. As discussed above, the ends 288, 289 and290, 291 of C-shaped seals 282, 284, respectively, bend inward, whichboth helps to funnel the platelet suspension through channels 286, 287and to minimize the amount of red blood cells that pass through channels286, 287. The point at which the red blood cells are near the entranceof channels 286, 287 may be monitored either visually or by a sensor, asdescribed below in detail. At this point the infusion of additionalaliquots of whole blood is terminated, and the second stage of thetwo-stage separation process begins.

During stage two of the separation process, the platelet suspensionwhich was pushed through channels 286, 287 into the second processingcompartment 293 flow under the influence of centrifugal force towardspositions within the second processing compartment 293 that have thegreatest radial distances, that is, towards concave wells 283, 285,where the platelets, being the higher density component of the plateletsuspension, begin to collect and pack. The platelets can then bewithdrawn from concave wells 283, 285 through outlet tube 278. In theabove-described two-stage process for the separation of a plateletsuspension from whole blood, the first and second C-shaped seals 282,284 thus serve as physical barriers between the red blood cells and theplatelets to facilitate the separation and collection of platelets fromwhole blood. First and second concave wells 283, 285 act as reservoirsfor containing the platelets as they are separated from the plateletsuspension in the second stage of the separation process.

After withdrawal of the platelets from the wells 283 and 285, inletlumen 230 is disconnected from the whole blood source container, afterwhich the remaining components in centrifuge bag 270 are evacuatedthrough inlet tube 276 by applying suction to inlet lumen 230 and aredirected to a disposable container. The inlet lumen 230 is thenreconnected to the whole blood source container, and the above-describedbatch process is repeated as many times as required until the desiredquantity of platelets has been harvested.

An alternative embodiment of a disposable, flexible centrifuge baghaving inner C-shaped seals is illustrated in FIG. 42 as centrifuge bag320. Disposable centrifuge bag 320 has a substantially flat, toroidal-or doughnut-shaped configuration having outer and inner perimeters 322and 324, respectively, and comprises radially extending upper and lowersheets 323, 325 formed from a substantially flexible material. The upperand lower sheets 323, 325 are superimposed and completely sealedtogether at outer perimeter 322 by an rf weld, heat weld or othercomparable method of adhering two surfaces. Inner perimeter 324 definescore 327 of bag 320.

Upper and lower sheets 323, 325 of centrifuge bag 320 are further sealedat two portions between the outer perimeter and the inner perimeter.That is, centrifuge bag 320 further comprises a first C-shaped seal 326located between the inner and outer perimeters 322, 324, and a secondC-shaped seal 328 located between the inner and outer perimeters 322,324. The first and second C-shaped seals 326, 328 have their concavesides facing each other such that when centrifuge bag 320 is viewed fromthe top as in FIG. 36, first and second C-shaped seals 326 and 328 aremirror images of each other. First and second C-shaped seals 326, 328together define an outer compartment 348 between the outer perimeter 322and first and second C-shaped seals 326 and 328, wherein the outercompartment 348 has a toroidal configuration. First and second C-shapedseals further define an compartment 350 between first and secondC-shaped seals 326, 328 and inner perimeter 324, wherein the innercompartment 350 has a doughnut shaped configuration. The ends 330 and332 of first C-shaped seal 326 are slightly curved inward towards theinner core 327, and likewise ends 334 and 336 of second C-shaped seal328 are slightly curved inward towards the inner core 327. The first andsecond C-shaped seals 326 and 328 are positioned such that ends 330 and334 of first and second C-shaped seals 326, 328, respectively, aredirectly opposite and spaced apart from each other, thereby definingfirst channel 335 therebetween, and such that ends 332 and 336 of firstand second seals 326 and 328, respectively, are directly opposite andspaced apart from each other, thereby defining second channel 337therebetween, wherein the first and second channels 335 and 337 arediametrically opposed. First and second channels 335 and 337 providefluid communication between the outer and inner compartments.

Centrifuge bag 320 further comprises an inlet port 340, in the lowersheet 325 for introducing fluid into outer compartment 348. Preferablythe inlet port 340 is spaced 90 degrees from channel 335 however itcould also be positioned at an angle greater or less than 90 degreesfrom channel 335. Centrifuge bag 320 further comprises first and secondoutlet ports 344, 346 in lower sheet 325 and positioned within channels335 and 337 for withdrawing a fluid compartment from centrifuge bag 320.

In a preferred embodiment, centrifuge bag 320 comprises inlet tube 338secured to the outside surface of upper sheet 323 or lower sheet 325 andradially extending from the center of core 327 towards the outerperimeter 322, wherein inlet tube 338 is fluidly connected at its distalend to inlet port 340. Inlet port 340 fluidly connects inlet tube 338with the outer chamber 348 of centrifuge bag 320. Inlet tube 338 isfluidly connected at its proximal end to umbilical cable 228, preferablyby an L-shaped connector (not shown). Further, in a preferred embodimentcentrifuge bag 320 comprises outlet tube 342 secured to the outsidesurface of upper sheet 323 or lower sheet 325 and extending across thediameter of core 327, wherein one end of outlet tube 342 is fluidlyconnected to first outlet port 344 and the other end of outlet tube 342is fluidly connected to second outlet port 346. Outlet tube is fluidlyconnected at its center to umbilical cable 228 via a T-shaped connector(not shown).

In an alternative embodiment of this invention, centrifuge bag 320comprises inlet tube 338 sandwiched between upper and lower sheets 323,325 and extending radially from the center of core 327 towards outerperimeter 322, wherein inlet tube 328 is fluidly connected at its distalend to inlet port 340, and outlet tube 342 sandwiched between upper andlower sheets 323, 325 and extending across the diameter of core 327,wherein one end of outlet tube 342 is fluidly connected to outlet port344 and the other end of outlet tube 342 is fluidly connected to outletport 346. When upper and lower sheets 323, 325 are sealed together atinner perimeter 324, inlet and outlet tubes 338, 342 are thereby sealedtherebetween. Inlet tube 338 and outlet tube 342 are fluidly connectedto umbilical cable 228 (not shown), which in this particular embodimentis a dual lumen tubing.

Centrifuge bag 320 is removably secured between rotor base 204 and rotorcover 206 of rotor 202 in a manner as described above so that centrifugebag 320 is held in a fixed position relative to rotor base 204 and rotorcover 206 during rotation of the centrifuge rotor 202. As will beappreciated by those of skill in the art, alternative embodiments ofrotor base 204 (FIG. 15) and rotor cover 206 (FIG. 16) as discussedabove with respect to centrifuge bag 270 will be required to accommodatethe design of centrifuge bag 370.

Centrifuge bag 370 may be used for the separation and isolation of oneor more components dissolved or suspended in a variety of fluid media,including, but not limited to, the separation of cellular componentsfrom biological fluids. For example, centrifuge bag 370 is useful forthe concentration and removal of platelets from whole blood. Therefore,the following description of the separation of platelets from wholeblood using centrifuge bag 320 is merely for purposes of illustrationand is not meant to be limiting of the use of bag 320. The separation ofa fluid medium such as whole blood in centrifuge bag 320 may beconsidered to be a one-stage separation process. With reference to FIG.36, centrifugation of whole blood begins with the introduction of analiquot of whole blood into centrifuge bag 320 through inlet port 340via inlet tube 338 during rotation of the centrifuge 20. Inlet tube 338is fluidly connected via inlet lumen 230 of umbilical cable 228 to ananticoagulated whole blood source. As the aliquot of whole blood entersthe outer chamber 348 of centrifuge bag 320, it quickly separatesradially within outer chamber 348 into various fractions based on thedensities of the components of the whole blood, including an outermostfraction containing the red blood cells which pack along the outerperimeter 322 of centrifuge bag 320, and inner fractions containing theplatelets and plasma. Continued infusion of whole blood adds anadditional volume of red blood cells and consequently pushes thefraction containing platelets inward. Additional infusions of wholeblood will continue to push the platelet-containing fraction furtherinward until the chamber 348 is substantially filled with red bloodcells (the remainder of the volume being plasma), such that theplatelet-containing fraction is pushed into channels 335, 337 and intothe vicinity of outlet ports 344, 346. As discussed above, the ends ofC-shaped seals 326, 328 curve slightly inward, which both helps tofunnel the platelet-containing fraction into channels 335, 337, and tominimize the amount of red blood cells that flow into channels 335, 337.The point at which the red blood cells are near the entrance of channels335, 337 may be monitored either visually or by a sensor, as describedbelow in detail. As the platelet-containing fraction enters the vicinityof outlet ports 344, 346, the infusion of whole blood is terminated, andsuction or other drawing means is applied to outlet tube 342 to withdrawthe platelet-containing fraction through outlet ports 344, 346.

After withdrawal of substantial portion of the platelet rich plasma,inlet lumen 230 is disconnected from the whole blood source containerand connected to a disposal container, after which the remainingcomponents in centrifuge bag 320 are evacuated through inlet port 34—byapplying suction to inlet tube 276 and are directed to a disposalcontainer. The inlet lumen 230 is then reconnected to the whole bloodsource container, and the above-described process is repeated as manytimes as required until the desired quantity of platelets has beenharvested.

B. Rigid Centrifuge Container

As can be appreciated, it may be desirable to maximize the surface areaof separated fraction to be harvested, since this maximizes the amountof the fraction which may be collected without increasing the potentialfor introducing impurities into the separation (e.g., adjacent, lighterdensity components may begin moving into the region of the fractionbeing harvested), and without increasing the size of the centrifuge toan undesirable degree.

In order to maximize the amount of the desired component (e.g., plateletrich plasma, white blood cells, or platelet poor plasma) which may beharvested, one embodiment of a centrifuge container of this inventionfor the separation of components in a fluid medium (e.g., whole blood),shown in FIGS. 45-52, is designed to position the desired component(e.g., platelet rich plasma) the platelet rich plasma at a region withinthe fixed centrifuge container or centrifuge bag so that the desiredfraction has a maximum horizontal surface area (i.e., width). Thus,another embodiment of this invention comprises a centrifuge container500 shown in FIG. 45. FIG. 45 is a side cross-sectional view of a rigidcontainer 500 comprising a rigid, annular body 510 having an axial core600 that is closed at the top end 610 and opened at the bottom end 620.Rigid container 500 further comprises an interior collection chamber 580for receiving and holding the fluid medium to be centrifuged and havingan outer perimeter 585 and an inner perimeter 590. The side,cross-sectional profile of chamber 580 is generally an off-centered“figure eight” or “dumbbell” shape, as shown in FIG. 46. As used herein,“figure eight” or “dumbbell” shaped means that the height of section Ais approximately equal to the height of section C, and the heights ofsections A and C are greater that the height of section B. Furthermore,as used herein, “off-center” means that the width W₁ from the center ofsection B to outer perimeter 585 is less than the width W₂ from thecenter of section B to inner perimeter 590 as shown in FIGS. 45 and 46.

Rigid container 500 further comprises inlet channel 550 extendingradially from core 600 to a point near the outer perimeter 585 and isfluidly connected at its distal end with the outer area of chamber 580.Rigid container 500 further comprises outlet channel 554 extendingradially from core 600 to the more central portion of chamber 580 (i.e.,the narrow portion or “neck” of the figure eight cross-section) and isfluidly connected at its distal end with chamber 580. While the inletand outlet channels 550, 554 are shown in FIG. 45 as being fluidlyconnected to the top end of chamber 580, the present invention alsoincludes embodiments wherein both channels 550, 554 are in fluidcommunication with the bottom end of chamber 580, or wherein channel 550is in fluid communication with the top end of chamber 580 and channel554 is in fluid communication with the bottom end of chamber 580, orvice versa. Inlet and outlet channels 550, 554 are fluidly connected todual lumen tubing 228 having an inlet lumen 230 and an outlet lumen 232.Rigid container 500 is removably secured to the upper surface 133 ofupper bearing assembly 130 with appropriate screws, fasteners or thelike (not shown). Inlet lumen 230 may be connected to a source for fluidmedium, and outlet lumen 232 may be connected to a suction means forwithdrawing the desired fraction from the chamber 580.

The configuration of chamber 580 is specifically designed to maximizethe collection of platelet rich plasma by centrifugation ofanticoagulated whole blood. More particularly, the shape of chamber 580increases the width of the platelet rich plasma fraction when viewedfrom the top and decreases the depth of the platelet rich plasmafraction when viewed from the side, thus allowing the withdrawal of agreater amount of platelet rich plasma. This unique design can be betterexplained by comparing FIGS. 44 and 47. FIG. 44 shows a side profile ofa rigid centrifuge container 500 as shown in FIG. 43, having a generallyoval profile and containing whole blood that has been separated intofour fractions by centrifugation. In FIG. 44, width W₃ indicates therelative horizontal width of the platelet rich plasma fraction to beharvested, and D₁ indicates the relative depth of the platelet richplasma fraction. FIG. 47 shows a side profile of rigid centrifugecontainer 580 of this invention having the above-described off-centeredfigure eight shape and containing whole blood that has been separatedinto four fractions by centrifugation. In FIG. 47, width W₄ indicatesthe relative horizontal width of the platelet rich plasma fraction 260to be harvested, and D₂ indicates the relative depth of the plateletrich plasma fraction 260. Width W₄ is necessarily wider than width W₃ inFIG. 44. Thus it can be easily appreciated that upon withdrawal of theplatelet rich plasma fraction 260 from the oval shaped container shownin FIG. 44, platelet poor plasma fraction 262 will shift closer to theoutlet tube 554 relatively quickly. In contrast the dumbbell shapedprofile of chamber 580 shown in FIG. 47 significantly increases thewidth W₄ while decreasing the average depth D₂, and therefore a greaterportion of the platelet rich plasma fraction 260 can be withdrawn withgreater accuracy before the platelet poor plasma fraction 262 reachesthe outlet tube 554.

In an embodiment where the platelet rich plasma is to be collected onecould design chamber 580 as follows. The configuration of chamber 580,that is, the relative heights A, B, and C as shown in FIG. 46, will bedetermined based on the typical location of the platelet rich plasmafraction 260 after centrifugation of whole blood. For example, in arigid centrifuge container 500 as illustrated in FIG. 45, having chamber580 with a 30 ml capacity and a radius of approximately 65 mm measuredfrom its rotational axis to the edge 630, the platelet rich plasma willcollect in chamber 580 at a region at a radial position ranging fromabout 35 to about 60 mm from the axis. In this region of the chamber580, as illustrated in FIG. 46, the chamber 580 has a height of about 10mm such that the horizontal surface area “B” of this region, illustratedin FIG. 46, is about 4 mm². Consequently, it can be appreciated thatbecause of the unique configuration of chamber 580, the surface area ofthe platelet rich plasma fraction 260 as illustrated in FIG. 47 may bemaximized without undesirably increasing the overall size of the rigidcentrifuge container 500. It will be appreciated by those skilled in theart that various geometric designs may be utilized depending on thefluid medium being centrifuged and the cellular fraction to becollected. The process for harvesting platelets from whole blood usingrigid container 500 may be achieved in a manner similar to thatdescribed for bag 226.

Rigid centrifuge container 500 may be made from any number of rigid,transparent materials that are capable of withstanding typicalsterilization conditions, including but not limited to acrylic resins,polycarbonate, or any clear thermal plastic. Preferably rigid container500 is made of a cost-effective material that is relatively inexpensiveto dispose of.

C. Centrifuge Rotor Having a Complex Interior Geometry

An alternate embodiment of a centrifuge rotor of this invention forholding flexible centrifuge bag 226 is illustrated in FIGS. 48-52.Generally and referring to FIGS. 48 and 49, the centrifuge rotor 755 isdefined by a rotor base 760 (FIGS. 48, 50 and 52) having a lower channel780, and a rotor cover 770 (FIGS. 49 and 51) having an upper channel782. The annular interior chamber 784 (FIG. 48) of rotor 755 is definedby lower and upper channels 780, 782, and has a generally off-centeredfigure eight side cross-sectional configuration specifically designed tomaximize the collection of platelet rich plasma by centrifugation ofanticoagulated whole blood, as discussed below in detail.

As illustrated in FIGS. 51 and 52, rotor base 760 comprises raisedannular rim 775 and raised column 786 which is axially disposed in theinterior of rotor base 760. Raised column 786 further has a groove 790(FIG. 52) extending the diameter of column 786. The height of rim 775 isequal to the height of column 786. As illustrated in FIG. 51, rotorcover 770 comprises raised annular rim 777 and raised column 788 whichis axially disposed in the interior of cover 770. Raised column 788further has a groove 792 (FIG. 52) extending the diameter of column 788.The height of rim 777 is equal to the height of column 788. Rotor base760 and rotor cover 770 are preferably made from any number of rigidtransparent materials including, but not limited to acrylic resins,polycarbonate, or any clear thermal plastic.

When centrifuge rotor 755 is to be assembled for use, flexible,doughnut-shaped centrifuge bag 226 having a center core 242 is placed inrotor base 760 such that center column 786 preferably, but notnecessarily, extends through the core of centrifuge bag 226, and inletand outlet tubes 248, 250 of bag 226 are seated in groove 790. Rotorcover 770 is superimposed on rotor base 760 such that grooves 790 and792 are aligned and further so that inlet and outlet tubes 248, 250 areseated in groove 792. In one embodiment, when cover 770 is appropriatelysecured to base 760 (e.g., with screws, clamps, or the like), rims 775and 777 are in complete contact with each other, and columns 786 and 788are preferably in complete contact with each other, thereby creatingchamber 784 (FIG. 48). Alternatively when cover 770 is secured to base760 as described, the inner perimeter of bag 226 is secured betweencolumns 786 and 788 such that the columns do not physically contact eachother.

When the generally flat, flexible centrifuge bag 226 is contained withinchamber 784 prior to the infusion of a fluid medium (e.g., whole blood),it will not fill the entire volume of chamber 784 but rather will have aradially extending, flat shape as centrifuge rotor 755 is spinning.However, after a sufficient volume of the fluid medium (e.g., wholeblood) has been introduced into flexible bag 226 through inlet tube 248such that bag 226 is substantially completely filled, it will beappreciated that filled centrifuge bag 226 will conform to the shape ofchamber 784 and consequently will have a off-centered figure eightshaped cross-section.

The off-centered figure eight configuration of the chamber 784 is ofapproximately the same configuration as the rigid bag 500. Therefore,for the same reasons, the shape of chamber 784 (and consequently theshape of filled bag 226), will assume an off-centered figure eight shapewherein the width of the platelet rich plasma fraction is greatlyincreased relative to the width of a filled bag having an ellipticalcross-sectional shape (see, for example, FIGS. 46 and 47).

As discussed above, a number of methods may be utilized to gauge theharvesting of the desired fraction (such as, but not limited to,platelet rich plasma) from the centrifuge bag. For instance, theseparation of platelet rich plasma fraction may be indicated by visualobservation of a concentric ring containing the platelet rich plasma(which will be a less colored fraction) and an outer red-coloredconcentric ring containing the red blood cells. In this case, when suchfraction(s) have been separated, the platelet rich plasma may bewithdrawn from centrifuge bag 226 by bent fitting 160 to direct theplatelet rich plasma to the appropriate collector.

As an alternative to the foregoing, sensors may be incorporated asdiscussed in detail below to detect the presence of the platelet richplasma fraction.

Based upon the foregoing, it can be appreciated that the centrifugalprocessing system 10 and the centrifuge rotors and bags of thisinvention have a plurality of features which are suited to harvestingplatelet rich plasma, white blood cells, platelet poor plasma or redblood cells from a patient's whole blood in accordance with each of theaspects of the present invention. For example, as discussed above,hematocrits (the volume of blood occupied by red blood cells, expressedas a percentage) vary from individual to individual. Thus, depending onthe amount of red blood cells present in a particular sample, the exactradial location of various blood components within the centrifuge bagafter centrifugation will also vary. The centrifuge bags of thisinvention overcome this issue by having an inlet tube capable of notonly introducing whole blood into the centrifuge bag, but also capableof withdrawing some of all of the red blood cell fraction as needed toshift the location of the fraction to be harvested into the area of theoutlet tube. Such features are presented in centrifuge bags 226, 270,320 and 500. Yet another embodiment of the centrifuge bags of thisinvention which overcomes problems with varying hematocrits iscentrifuge bag 226′ having multiple outlet tubes.

Additionally, the centrifugal processing system 10 effectively providesa closed system which enhances the potential for maintaining a desireddegree of sterility associated with the entire procedure since materialscan thus be both provided to and removed from the centrifuge bag duringrotation of the centrifuge via, for instance, a dual lumen tubingconnected to a fluid source (e.g., anticoagulated whole blood withdrawnfrom a patient before or during surgery) and collection containers(i.e., for the preparation of a platelet gel), without interrupting theprocess, and thus without significant exposure of the materials toenvironmental conditions.

Moreover, the portable size of the centrifugal processing system 10 incombination with the above-described features of shifting the separatedfractions and maximizing the surface area of the harvested fractionallows for increased processing capabilities autologous platelet gelover larger, conventional centrifuges.

The on-line harvesting capabilities of the centrifugal processing system10 allows for continuous, dynamic separation and collection of plateletrich plasma, white blood cells, red blood cells and platelet poorplasma, by adjusting the input and removal of fluid medium and separatedfractions as described above. Further, the orientation of the flexibleand rigid centrifuge bags of this invention and of the contents therein(e.g., being generally radially extending) is not significantly modifiedin the transformation from separation to harvesting of the variousconstituents. Moreover, vortexing throughout the contents of thecentrifuge bags of this invention is reduced or eliminated since thecentrifugal processing system 10 does not have to be decelerated orstopped for addition of fluid medium or removal of the various fractionstherefrom.

Further, the general orientation of the flexible and rigid centrifugebags of the invention (e.g., substantially horizontal) is maintainedduring removal of the desired whole blood fraction similar to theorientation of the centrifuge bags assumed during centrifugation tofurther assist in maintaining the degree of separation provided bycentrifugation. Consequently, the potential is reduced for disturbingthe fractions to the degree where the separation achieved is adverselyaffected.

Although the present invention has been described with regard to theseparation of whole blood components, it will be appreciated that themethods and apparatus described herein may be used in the separationcomponents of other fluid media, including, but not limited to wholeblood with density gradient media; cellular components, or sub-sets ofthe four whole blood components previously defined.

While blood separation and materials handling may be manuallycontrolled, as discussed above, a further embodiment of the presentinvention provides for the automation of at least portions of theseparation and material handling processes. Referring to FIG. 53, anautomated centrifugal processing system 800 is illustrated that isgenerally configured to provide automated control over the steps ofinputting blood, separating desired components, and outputting theseparated components. The following discussion of the processing system800 provides examples of separating platelets in a blood sample, but theprocessing system 800 provides features that would be useful forseparating other components or fractions from blood or other fluids.These other uses for the processing system 800 are considered within thebreadth of this disclosure. Similarly, the specific components discussedfor use in the processing system 800 are provided for illustrationpurposes and not as limitations, with alternative devices being readilyapparent to those skilled in the medical device arts.

In the embodiment illustrated in FIG. 53, the processing system 800includes a blood source 802 connected with a fluid line 804 to an inletpump 810. A valve 806, such as a solenoid-operated valve or a one-waycheck valve, is provided in the fluid line 804 to allow control of flowto and from the blood source 802 during operation of the inlet pump 810.The inlet pump 810 is operable to pump blood from the blood source 802through the fluid line 818 to a centrifuge 820. Once all or a selectportion of the blood in the blood source 802 have been pumped to a bloodreservoir 824 of the centrifuge 820 the inlet pump 810 is turned off andthe blood source 802 isolated with valve 806. The inlet pump 810 may beoperated at later times to provide additional blood during the operationof the processing system 800 (such as during or after the removal of aseparated component).

The centrifuge 20 preferably includes a flexible centrifuge bag, forexample 226, 226′, 270, or 320, positioned within the rotor 202 forcollecting the input blood, or alternatively rotor 202 may be a rigidcontainer having an off centered figure eight shaped chamber, which maycollect blood directly as discussed previously. Thus, while theembodiment described below illustrates a centrifuge having bag 226, itis to be understood that the alternative centrifuge bags disclosedherein may be used in a similar manner. The centrifuge 20 as discussedabove has an internal mid-shaft gear assembly 108 that provides themotive force to rotate the rotor assembly 200, and particularly therotor 202, at a rotation rate that is adequate to create centrifugalforces that act to separate the various constituents or components ofthe blood in the rotor 202. The drive assembly 822 may comprise a numberof devices useful for generating the motive force, such as an electricmotor with a drive shaft connected to internal drive components of thecentrifuge 20. In a preferred embodiment, the drive assembly 822comprises an electric motor that drives a belt attached to an exteriorportion of the centrifuge 20 and more particularly to the timing beltring 44. To obtain adequate separation, the rotation rate is typicallybetween about 0 RPM and 5000 RPM, and in one embodiment of theinvention, is maintained between about 0 RPM and 5000 RPM.

As discussed in detail previously, components of particular densitiesassume radial positions or belts at differing distances from the centralaxis A of the rotor 202. For example, the heavier red blood cellstypically separate in an outer region while lower density plateletsseparate into a region more proximal to the central axis of the rotor202. Between each of these component regions, there is an interface atwhich the fluid density measurably changes from a higher to a lowerdensity (i.e., as density is measured from an outer to an inner region),and this density interface is used in some embodiments of thecentrifugal processing system 10 to identify the location of componentregions (as will be discussed in more detail below). In a preferredembodiment, the drive assembly 822 continues to operate to rotate thecentrifuge 20 to retain the separation of the components throughout theoperation of the centrifugal processing system 10.

Once blood separation has been achieved within the rotor 202, the outletpump 830 is operated to pump select components from the rotor 202through outlet lumen 828. As discussed previously, in relation to thefeatures of the disposable blood centrifuge bag 226, the centrifuge bagheld within the rotor 202 preferably is configured to allow theselective removal of a separated blood component, such as plateletslocated in a platelet rich plasma region, by the positioning of anoutlet lumen 232 a radial distance from the central axis of thecentrifuge bag 226. Preferably, this radial distance or radial locationfor the outlet lumen is selected to coincide with the radial location ofthe desired, separated component or the anticipated location of theseparated component. In this manner, the outlet pump 830 only (orsubstantially only) removes a particular component (such as plateletsinto container 400) existing at that radial distance. Once all or adesired quantity of the particular component is removed from thecentrifuge bag 226, operation of the outlet pump 830 is stopped, and anew separation process can be initiated. Alternatively, in a preferredembodiment, additional blood is pumped into the centrifuge by 226 byfurther operating the inlet pump 810 after or concurrent with operationof the outlet pump 830.

A concern with fixing the radial distance or location of the outlet portis that each blood sample may have varying levels or quantities ofdifferent components. Thus, upon separation, the radial distance orlocation of a particular component or component region within thecentrifuge bag 226 varies, at least slightly, with each different bloodsample. Additionally, because of the varying levels of components, thesize of the component region also varies and the amount that can bepumped out of the centrifuge bag 226 by the outlet pump 830 withoutinclusion of other components varies with each blood sample. Further,the position of the component region will vary in embodiments of theseparation system 10 in which additional blood is added after or duringthe removal of blood by the outlet pump 830.

To address the varying location of a particular separated component, thecentrifugal processing system 10 preferably is configured to adjust thelocation of a separated component to substantially align the radiallocation of the separated component with the radial location of theoutlet port. For example, the centrifugal processing system 10 may beutilized to collect platelets from a blood sample. In this example, thecentrifugal processing system 10 preferably includes a red blood cellcollector 812 connected to the inlet pump 810 via fluid line 814 havingan isolation valve 816 (e.g., a solenoid-operated valve or one-way checkvalve). Alternatively, the pump or syringe may also act as the valve.The inlet pump 810 is configured to selectively pump fluids in twodirections, to and away from the centrifuge 820 through fluid line 818,and in this regard, may be a reversible-direction peristaltic pump orother two-directional pump. Similarly, although shown schematically withtwo fluid lines 804 and 814, a single fluid line may be utilized as aninlet and an outlet line to practice the invention.

Operation of the inlet pump 810 to remove fluid from the centrifuge bag226 is useful to align the radial location of the desired separatedcomponent with the outlet tube 250 and inlet tube 248 of the centrifugebag 226. When it is desired to align platelets or platelet rich plasmawith the outlet tube 250, the inlet tube 248 connected to lumen 232 and230, respectively, inlet tube 248 is preferably at a greater radialdistance than the outlet tube 250. When suction is applied to the inletlumen 230 by inlet pump 810, red blood cells are pumped out of thecentrifuge bag 226 and into the red blood cell collector 812. As redblood cells are removed, the separated platelets (i.e., the desiredcomponent region) move radially outward to a new location within thecentrifuge bag 226. The inlet pump 810 is operated until the radialdistance of the separated platelets or platelet region from the centralaxis is increased to coincide with the radial distance or location ofthe outlet tube 250 of the centrifuge bag 226. Once substantialalignment of the desired component region and the outlet tube 250 isachieved, the outlet pump 830 is operated to remove all or a selectquantity of the components in the aligned component region.

To provide automation features of the invention, the centrifugalprocessing system 10 includes a controller 850 for monitoring andcontrolling operation of the inlet pump 810, the centrifuge 20, thedrive assembly 822, and the outlet pump 830. Numerous control devicesmay be utilized within the centrifugal processing system 10 toeffectively monitor and control automated operations. In one embodiment,the controller 850 comprises a computer with a central processing unit(CPU) with a digital signal processor, memory, an input/output (I/O)interface for receiving input and feedback signals and for transmittingcontrol signals, and software or programming applications for processinginput signals and generating control signals (with or without signalconditioners and/or amplifiers). The controller 850 is communicativelylinked to the devices of the centrifugal processing system 10 withsignal lines 860, 862, 864, 866, and 868 which may include signalconditioning devices and other devices to provide for propercommunications between the controller 850 and the components of thecentrifugal processing system 10.

Once blood is supplied to the blood source container 802, the operatorpushes the start button and the controller 850 transmits a controlsignal over signal line 864 to the drive assembly 822, which may includea motor controller, to begin rotating the centrifuge 20 to cause thecomponents of the blood in centrifuge bag 226 to separate intoradially-positioned regions (such as platelet rich plasma regions).After initiation of the centrifuge spinning or concurrently withoperation of the drive assembly 822, the controller 850 generates acontrol signal over signal line 860 to the inlet pump 810 to beginpumping blood from the blood source container 802 to the centrifuge bag226 of the centrifuge 20. In some embodiments of the processing system800, the drive assembly 822 is operable at more than one speed or over arange of speeds. Additionally, even with a single speed drive shaft therotation rate achieved at the centrifuge 20 may vary. To address thisissue, the processing system 10 may include a velocity detector 858 thatat least periodically detects movement of the centrifuge bag 226 portionof the centrifuge 20 and transmits a feedback signal over signal line866 to the controller 850. The controller 850 processes the receivedsignal to calculate the rotation rate of the centrifuge 20, and ifapplicable, transmits a control signal to the drive assembly 822 toincrease or decrease its operating speed to obtain a desired rotationrate at the centrifuge bag 226.

To determine when separation of the components in the centrifuge bag 226is achieved, the processing system 800 may be calibrated to account forvariations in the centrifuge 20 and drive assembly 822 configuration todetermine a minimum rotation time to obtain a desired level of componentseparation. In this embodiment, the controller 850 preferably includes atimer mechanism 856 that operates to measure the period of time that thecentrifuge 20 has been rotated by the drive assembly 822 (such as bybeginning measuring from the transmission of the control signal by thecontroller 850 to the drive assembly 822). When the measured rotationtime equals the calibrated rotation time for a particular centrifuge 20and drive assembly 822 configuration, the timing mechanism 856 informsthe controller 850 that separation has been achieved in the centrifugebag 226. At this point, the controller 850 operates to transmit controlsignal over signal line 860 to the input pump 810 to cease operation andto the outlet pump 830 over signal line 868 to initiate operation topump a separated component in the component region adjacent the outletport of lumen 232 of centrifuge bag 226 through fluid line 828. Inanother embodiment where rotation time is utilized by controller 850,the velocity feedback signal from the velocity detector 858 is utilizedby the controller 850 to adjust the rotation time as necessary to obtainthe desired level of component separation. For example, the centrifugalprocessing system 10 can be calibrated for a number of rotation ratesand the corresponding minimum rotation times can be stored in a look uptable for retrieval by the controller 850 based on a calculated rotationrate. Rotational rates may be varied either manually or automatically tooptimize cellular component position and or concentration.

Because the location of component separation regions varies duringseparation operations, a preferred embodiment of the centrifugalprocessing system 800 includes a sensor assembly 840 to monitor theseparation of components within the centrifuge bag and to transmitfeedback signals over line 862 to the controller 850. As will beunderstood by those skilled in the art, numerous sensor devices existfor detecting the presence of certain components in a fluid, andspecifically a blood, sample. Many of these devices comprise a source ofradiant energy, such as infrared, laser, or incandescent light, and acompatible radiant energy-sensitive detector that reacts to the receivedenergy by generating an electric signal. Briefly, these radiant energydevices are useful because the detected signal varies in a measurablefashion with variances in the density of the material through whichbeams of the radiant energy are passed. According to the invention, thesensor assembly 840 may comprise any of these well-known types ofradiant energy source and detector devices and other sensor devicesuseful for measuring the existence of constituents of fluids such asblood.

The source and the detector of the sensor assembly 840 are preferablylocated within the centrifugal processing system 800 to allow monitoringof the centrifuge bag 226 and, particularly, to identify the presence ofa particular blood component in a radial position coinciding with theradial position of the outlet port of the centrifuge bag 226. In oneembodiment, the radiation beams from the source are transmitted througha “window” in the centrifuge bag 226 that has a radial location that atleast partially overlaps the radial location of the outlet port. Duringoperation of the centrifugal processing system 800, the feedback signalsfrom the detector of the sensor assembly 840 allow the controller 850 toidentify when a density interface has entered the window. This may occurfor a number of reasons. When red blood cells are being removed byoperation of the inlet pump 810 to remove fluid from the centrifuge bag226 via the inlet tube 248. The change in density may also occur when adenser component is being added to the centrifuge bag 226 causing theparticular blood component to be pushed radially inward. In thecentrifugation of whole blood, this occurs when additional blood isadded by operation of the input pump 810 and red blood cells collect ina region radially outward from the platelet region.

To account for differing movement of the density interface, the windowof the radiation source may be alternatively positioned radially inwardfrom the location of the outlet tube 250 of the centrifuge bag 226. Bypositioning the window inward from the outlet tube 250, the controller850 can identify when the outlet pump 830 has nearly removed all of theparticular component of the monitored region and/or when the inlet pump810 has removed a quantity of denser components causing the monitoredregion to move radially outward. The controller 850 can then operate tosend control signals to turn off the outlet pump 830 or the inlet pump810 (as appropriate) to minimize the amount of undesired components(lower density components) that enter the outlet tube 250.Alternatively, the sensor assembly 840 may have two radiation sourcesand detectors, and the second window of the sensor assembly 840 may belocated a distance radially outward from the outlet tube 250. With twosensing windows, the sensor assembly 840 is operable to provide thecontroller 850 information about a density interface moving radiallyinward toward the outlet tube 250 (such as when red blood cells areadded). In response, the controller 850 can generate a control signal tothe inlet pump 810 to operate to pump the denser components, such as redblood cells, out of the centrifuge bag 226. Two sensing windows alsoallow the controller 850 to detect a density interface moving outward,which allows the controller 850 to shut off the outlet pump 830 (and/orthe inlet pump 810 to stop evacuating processes) and/or to start theinlet pump 810 to add additional blood.

To further clarify operation of the processing system 800, FIG. 54 isprovided which illustrates the timing and relationship of controlsignals generated by the controller 850 and the receipt of feedbacksignals from the sensor assembly 840. In this embodiment, the radiationdetector of the sensor assembly 840 is positioned adjacent outlet tube(inlet to the outlet pump 830) in the centrifuge bag 226 to sensedensity changes in the fluid flowing past the outlet tube 250. Asillustrated, operation of the processing system 800 begins at time t₀,with the inlet pump 810, the outlet pump 830, and the centrifuge driveassembly 822 all being off or not operating. At time t₁, the controller850 operates in response to operator input or upon sensing the bloodsource 802 is adequately filled (sensor not shown) to generate a controlsignal on line 864 to begin operating the centrifuge drive assembly 822to rotate the centrifuge bag 226. In some embodiments, this controlsignal over line 864 also contains rotation rate information toinitially set the operating speed of the drive assembly 822.Concurrently or at a selected delay time, the controller 850 generates acontrol signal on line 860 to start the inlet pump 810 in aconfiguration to pump fluid to the centrifuge bag 226 over fluid line818. The sensor assembly 840 provides an initial density feedback signalto the controller 850 on line 862, which the controller 850 can processto determine an initial or unseparated density adjacent the outlet tube.Alternatively, the controller 850 may be configured to request afeedback signal from the sensor assembly 840 after a set delay period(as measured by the timer mechanism 856) to allow separation of thecomponents being pumped into the centrifuge bag 226 (such as thecalibrated, minimum rotation time discussed above) into regions.

At time t₂, the controller 850 functions to align the region having thedesired density, such as a region comprising a higher density ofplatelets, adjacent the detector of the sensor assembly 840 (i.e.,adjacent the outlet tube). To achieve alignment, the controller 850transmits a control signal over line 860 to the inlet pump 810 to stoppumping fluid to the centrifuge bag 226, to reverse pumping directionsincluding shutting valve 806 and opening valve 816, and to begin pumpingcomponents having a higher density then the particular, desiredcomponent from the centrifuge bag 226 to the collector 812. For example,when the centrifugal processing system 10 is operated to separate andcollect platelets or platelet rich plasma, the inlet pump 810 at time,t₂, is operated to pump out the red blood cell fraction by applyingsuction at the inlet tube 248 to the centrifuge bag 226. At time t₃, thedensity of the fluid adjacent the outlet tube 250 begins to change asdenser components are removed by the inlet pump 810, and the sensorfeedback signal being transmitted to the controller 850 changes inmagnitude. The sensor feedback signal continues to change in magnitude(either becoming stronger or weaker depending on the particular sensorutilized and the material being collected) until at time t₄, when thecontroller 850 processes the feedback signal and determines that thedensity of the adjacent fluids is within a desired range. Thistransition can also be thought of as detecting when an interface betweentwo regions of differing densities passes by the location of thedetector of the sensor assembly 840.

With the region of the desired, separated component aligned with theoutlet tube 250, the controller 850 operates at time t₄, to send acontrol signal over line 860 to stop operations of the inlet pump 810.Also, at time t₄, or at any time thereafter, the controller 850generates a control signal over line 868 to begin operation the outletpump 830 to apply suction at the outlet tube 250 of the centrifuge bag226 to remove the desired component, such as the platelet rich plasmafraction, from the centrifuge bag 226. At time t₅, the sensor feedbacksignal again begins to change in magnitude as the density of the fluidnear the outlet tube 250 begins to change, such as when platelet poorplasma begins to enter the sampling window of the sensor assembly 840.At time t₆, the density of the fluid adjacent the outlet tube 250 and,hence, in the sampling window is outside of a desired density range(e.g., the fluid has less than a predetermined percentage of plateletsor other desired fluid component). In response, the controller 850transmits a control signal on line 868 to halt operations of the outletpump 830. Of course, the controller 850 can be operated to transmit thesignal to the outlet pump 830 at any time prior to time t₆, such as at atime after time t₅, when the density of the adjacent fluid begins tochange but prior to time t₆ or based on volume removed. The controller850 can then operate any time after time t₆, to halt operation of thecentrifuge drive assembly 822. Further, as discussed above, operationsof the separation centrifugal processing system 800 can be repeated withthe inlet pump 810 being operated to add additional fluid, e.g., blood,after time t₆. Alternatively, the inlet pump 810 and the outlet pump 830may be operated concurrently to add an additional volume of blood with acorresponding new amount of the component being collected after time t₄,to extend the period of time between detection of the interface at timet₄ and the detection of an out of range density at time t₆.

In the above discussion of the automated processing system 800, a sensorassembly 840 was shown in FIG. 53 schematically, and it was noted thatthe location of a radiant energy source and a detector may be anylocation within the processing system 800 useful for obtaining anaccurate measurement of separating blood components within thecentrifuge bag 226. For example, the source and detector can be bothpositioned within the centrifuge 20 at a location adjacent thecentrifuge bag 226. In this embodiment, problems may arise withproviding proper signal and power line connections to the source andsensor and with accounting for the rotation of the centrifuge andportions of the sensor assembly 840. Hence, one preferred embodiment ofthe processing system 800 provides for an externally positioned sensorassembly 840 including source and detector to simplify the structure ofthe centrifuge 20 while still providing effective density determinationsof fluids within the blood reservoir.

FIG. 55 illustrates a general side view of the relevant components ofthis external sensor embodiment of the centrifugal processing system800. Generally, the centrifuge 20 comprises a rotor extension portion880 and a drive portion 881, which is connected to the drive assembly822 (connection not shown). Both the centrifuge 20 and the rotorextension portion 880 rotate about a central or rotation axis, c_(axis),of the centrifuge 20. As discussed in more detail with respect to theinternal gearing features of the centrifuge 20, the drive portion 881spins in a ratio of 2 to 1 (or other suitable ratio) relative to thereservoir extension portion 880 to control twisting of inlet and outletfluid lines to the rotor extension portion 880. The internal gearingfeatures of the centrifuge 20 also enable the centrifuge 20 toeffectively obtain rotation rates that force the separation ofcomponents with differing densities while limiting the risk that densercomponents, such as red blood cells, will become too tightly packedduring separation forming a solid, dense material that is more difficultto pump or remove from the centrifuge 20.

Referring again to FIG. 55, the rotor extension portion 880 is shownlocated on the upper end of the centrifuge 20 and includes a centrifugebag 226 or other receptacle. Preferably, the rotor extension portion 880is fabricated from a transparent or partially transparent material, suchas any of a number of plastics, to allow sensing of fluid densities. Therotor extension portion 880 extends a distance, d_(over), beyond theouter edge of the centrifuge 20 as measured radially outward from thecentral axis, c_(axis). The distance, d_(over), is preferably selectedsuch that the desired component, such as the platelet rich plasmafraction, to be collected readily separates into a region at a pointwithin the centrifuge bag 226 that also extends outward from thecentrifuge 20. In this regard, the rotor extension portion 880 is alsoconfigured so that the centrifuge bag 226 extends within the rotorextension portion 880 to a point near the outer circumference of therotor extension portion 880. The distance, d_(over), selected forextending the rotor extension portion 880 is preferably selected tofacilitate alignment process (discussed above) and to control the needfor operating the input pump 810 to remove denser components. In oneembodiment, the distance, d_(over), is selected such that duringseparation of a typical blood sample center of the platelet rich regionis about one half the extension distance, d_(over), from thecircumferential edge of the centrifuge 20.

The sensor assembly 840 is entirely external to the centrifuge 20 asshown in FIG. 55. The sensor assembly 840 includes a source 882 foremitting beams 884 of radiant energy into and through the rotorextension portion 880 and the included centrifuge bag 226. Again, asdiscussed previously, the radiant energy source 882 may be nearly anysource of radiant energy (such as incandescent light, a strobe light, aninfrared light, laser and the like) useful in a fluid density sensor andthe particular type of detector or energy used is not as important asthe external location of the source 882. The sensor assembly 840 furtherincludes a detector 886 that receives or senses beams 888 that havepassed through the centrifuge bag 226 and have impinged upon thedetector 886. The detector 886 is selected to be compatible with thesource 882 and to transmit a feedback signal in response sensing theenergy beams 888. The detector 886 (in combination with the controller850 and its processing capacities) is useful for detecting the densityof fluids in the centrifuge bag 226 between the source 882 and thedetector 886. Particularly, the sensor assembly 840 is useful foridentifying changes in fluid density and interfaces between fluids withdiffering densities. For example, the interface between a regioncontaining separated red blood cells and a region containing theplatelet rich plasma fraction, and the interface between the plateletrich plasma region and a platelet-poor plasma region.

With some source and detector configurations, a sampling window iscreated rather than a single sampling point (although a single samplingpoint configuration is useful as part of the invention as creating awindow defined by a single radial distance). The sampling window isdefined by an outer radial distance, d_(OUT), from the central axis,c_(axis) and an inner radial distance, d_(IN). As may be appreciated,for many source and detector configurations the size of the samplingwindow may be rather small approximating a point and may, of course varyin cross-sectional shape (e.g., circular, square, rectangular, and thelike). As discussed previously, it is preferable that the sensorassembly 840 be positioned relative to the reservoir extension portion880 and the centrifuge bag 226 such that the sampling window created bythe source 882 and detector 886 at least partially overlaps the radialposition of the region created during separation processes containing acomponent of particular density, such as platelets. This may be acalibrated position determined through calibration processes of thecentrifuge 20 in which a number of blood (or other fluid) samples arefully separated and radial distances to a particular region aremeasured. The determined or calibrated position can then be utilized asa initial, fixed location for the sensor assembly 840 with the source882 and detector 886 being positioned relative to the rotor extensionportion 880 such that the sampling window overlaps the anticipatedposition of the selected separation region. Of course, each sample mayvary in content of various components which may cause this initialalignment to be inaccurate and operations of the centrifugal processingsystem 800 may cause misalignment or movement of regions. Hence,alignment processes discussed above preferably are utilized in additionto the initial positioning of the sampling window created by the sensorassembly 840.

In an alternate embodiment, the sensor assembly 840 is not in a fixedposition within the separation system 800 and can be positioned duringseparation operations. For example, the sensor assembly 840 may bemounted on a base which can be slid radially inward toward thecentrifuge 20 and radially outward away from the centrifuge 20 to varythe distances, d_(IN) and d_(OUT). This sliding movement is useful forproviding access to the centrifuge bag 226, such as to insert and removea disposable bag. During operation, the sensor assembly 840 wouldinitially be pushed outward from the centrifuge 20 until a new bag wasinserted into the centrifuge bag 226. The sensor assembly 840 could thenbe slid inward (or otherwise moved inward) to a calibrated position.Alternatively, the centrifugal processing system 800 could be operatedfor a period of time to achieve partial or full separation (based on atimed period or simple visual observation) and then the sensor assembly840 slid inward to a position that the operator of the centrifugalprocessing system 800 visually approximates as aligning the samplingwindow with a desired region of separated components (such as theplatelet rich plasma region). The effectiveness of such alignment couldthen readily be verified by operating the sensor assembly 840 to detectthe density of the fluids in the centrifuge bag 226 and a calculateddensity (or other information) could be output or displayed by thecontroller 850. This alternate embodiment provides a readilymaintainable centrifugal processing system 800 while providing thebenefits of a fixed position sensor assembly 840 and added benefits ofallowing easy relative positioning to obtain or at least approximate adesired sample window and separation region alignment.

In some situations, it may be preferable to not have a rotor extensionportion 880 or to modify the rotor extension portion 880 and the sensorassembly 840 such that the extension is not significant to monitoringthe separation within the blood reservoir or centrifuge bag 226. Twoalternative embodiments or arrangements are illustrated in FIGS. 56 and57 that provide the advantages of an external sensor assembly 840 (suchas an external radiation source and detector). With these furtherembodiments provided, numerous other expansions of the discussed use ofan external sensor will become apparent to those skilled in the arts andare considered within the breadth of this invention.

Referring to FIG. 56, a rotor 202 is illustrated that has no extendingportion (although some extension may be utilized) and contains thecentrifuge bag 226. Again, the rotor 202 and centrifuge bag 226 arepreferably fabricated from plastics or other materials that allowradiation to pass through to detect changes in densities or otherproperties of fluid samples within the centrifuge bag 226. In thisembodiment of the sensor assembly 840, the radiation source 882 and thedetector 886 are not positioned on opposing sides of the rotor 202.Instead, a reflector 885 (such as a mirror and the like) is positionedwithin the drive portion 881 of the centrifuge to receive the radiationbeams 884 from the radiation source 882 and direct them through theportion 880 and centrifuge bag 226. The detector 886 is positionedwithin the sensor assembly 840 and relative to the centrifuge 20 toreceive the deflected or reflected beams 888 that have passed throughthe fluid sample in the centrifuge bag 226. In this manner, the samplingwindow within the centrifuge bag 226 can be selected to align with theanticipated location of the fraction that is to be collected uponseparation. In a preferred embodiment, the sampling window at leastpartially overlaps with the location of the outlet tube of the bloodreservoir or centrifuge bag 226.

In one embodiment, the drive portion is fabricated from anon-transparent material and a path for the beams 884 from the radiationsource 884 to the reflector 885 is provided. The path in one preferredembodiment is an opening or hole such as port 154 or 156 (FIG. 14) inthe side of the drive portion 881 that creates a path or tunnel throughwhich the beams 884 travel unimpeded. Of course, the opening may bereplaced with a path of transparent material to allow the beams totravel to the reflector 885 while also providing a protective cover forthe internals of the drive portion 881. A path is also provideddownstream of the reflector 885 to allow the beams 884 to travel throughthe drive portion 881 internals without or with minimal degradation.Again, the path may be an opening or tunnel through the drive portionleading to the portion 202 or be a path created with transparentmaterials. The beams 884 in these tunnel path embodiments enter thedrive portion 881 one time per revolution of the drive portion 881,which provides an acceptable rate of sampling. Alternatively, areflector 885 may readily be provided that extends circumferentiallyabout the center axis of the drive portion 881 to provide a samplingrate equivalent to the rate of beam 884 transmission. Of course, thepositions of the radiation source 882 and the detector 886 may bereversed and the angle of the reflector 885 and transmission of thebeams 884 may be altered from those shown to practice the invention.

A further embodiment of an external sensor assembly 840 is provided inFIG. 57. In this embodiment, the radiation source 882 also acts as aradiation detector so there is no need for a separate detector. In thismore compact external sensor configuration, the radiation source anddetector 882 transmits beams 884 into the rotating drive portion 881through or over the path in the drive portion 881. The reflector 885reflects the beams 884 toward the rotor 202 and the centrifuge bag 226to create a sampling window within the centrifuge bag 226 in whichdensity changes may be monitored. After passing through the centrifugebag 226 and included fluid sample, the beams 888 strike a secondreflector 887 that is positioned within the rotor 202 to reflect thebeams 888 back over the same or substantially the same path through thecentrifuge bag 226 to again strike the reflector 885. The reflector 885directs the beams 888 out of the drive portion 881 and back to theradiation source and detector 882 which, in response to the impingingbeams 888, transmits a feedback signal to the controller 850 for furtherprocessing.

In one embodiment, the beams 884 enter the driving portion 881 onceduring every revolution of the driving portion 881. The portion 880 ispreferably rotating twice for every rotation of the driving portion 881,as discussed in detail above, and hence, the second reflector 887 isaligned to receive the beams 888 only on every other rotation of thedriving portion 881. Alternatively, a pair of reflectors 887 may bepositioned in the rotor 202 such that the beams 888 may be received andreflected back through the centrifuge bag 226 once for every rotation ofthe driving portion 881. In yet a further embodiment, the reflector 885and second reflector 887 may expand partially or fully about the centeraxis of the centrifuge 20 (with corresponding openings and/ortransparent paths in the driving portion 881) to provide a highersampling rate.

According to an important feature of the invention, temperature controlfeatures are provided in an alternate embodiment of the automatedprocessing system invention 900, as illustrated in FIG. 58. Providingtemperature controls within the processing system 900 can take manyforms such as controlling the temperature of input fluid samples fromthe blood source 802, monitoring and controlling the temperature offluids in the centrifuge bag 226 to facilitate separation processes, andcontrolling the operating temperature of temperature sensitivecomponents of the processing system 900. These components include butare not limited to, red blood cells, white blood cells, plasma, plateletrich plasma or any of these components mixed with other drugs, proteinsor compounds. In a preferred embodiment of the invention, a temperaturecontrol system is included in the processing system 900 to heatcomponents removed from the centrifuge bag 226 by the outlet pump 830 toa desired temperature range. For example, when the processing system 900is utilized in the creation of autologous platelet gel, a dispenserassembly 902 is included in the processing system 900 and includeschambers or syringes for collecting and processing platelet rich plasmadrawn from the centrifuge 20. As part of the gel creation process, it istypically desirable to activate the platelets in the harvested plateletrich plasma fraction prior to the use of the gel (e.g., delivery to apatient). The temperature control system is useful in this regard forraising, and for then maintaining, the temperature of the platelets inthe dispenser assembly to a predetermined activation temperature range.In one embodiment of the gel creation process, the activationtemperature range is 25° C. to 50° C. and preferably 37° C. to 40° C.,but it will be understood that differing temperature ranges may readilybe utilized to practice the invention depending on the desiredactivation levels and particular products being processed or createdwith the processing system 900.

Referring to FIG. 58, the temperature control system of the processingsystem 900 includes a temperature controller 904 that is communicativelylinked to the controller 850 with feedback signal line 906. Thecontroller 850 may be utilized to initially set operating temperatureranges (e.g., an activation temperature range) and communicate thesesettings over feedback signal line 906 to the temperature controller904. Alternatively, the temperature controller 904 may includeinput/output (I/O) devices for accepting the operating temperatureranges from an operator or these ranges may be preset as part of theinitial fabrication and assembly of the processing system 900. Thetemperature controller 904 may comprise an electronic control circuitallowing linear, proportional, or other control over temperatures andheater elements and the like. In a preferred embodiment, the temperaturecontroller 904 includes a microprocessor for calculating sensedtemperatures, memory for storing temperature and control algorithms andprograms, and I/O portions for receiving feedback signals from thermosensors and for generating and transmitting control signals to varioustemperature control devices (e.g., resistive heat elements, fan rotors,and other devices well-known to those skilled in the heating and coolingarts).

As illustrated, a temperature sensor 908 comprising one or moretemperature sensing elements is provided to sense the temperature of thedispenser assembly 902 and to provide a corresponding temperaturefeedback signal to the temperature controller 904 over signal line 910(such as an electric signal proportional to sensed temperature changes).The temperature sensor 908 may be any temperature sensitive deviceuseful for sensing temperature and, in response, generating a feedbacksignal useful by the temperature controller 904, such as a thermistor,thermocouple, and the like. In a preferred embodiment, the temperaturesensor 908 is positioned within the dispenser assembly 902 to be in heattransferring or heat sensing contact with the syringes or other chamberscontaining the separated product which is to be activated. In thismanner, the temperature controller 904 is able to better monitor whetherthe temperature of the relevant chambers within the dispenser assembly902 is within the desired activation temperature range.

To maintain the chambers of the dispenser assembly 902 within atemperature range, a heater element 913 is included in the temperaturecontrol system and is selectively operable by the temperature controller904 such as by operation of a power source based on signals receivedfrom the temperature sensor 908. The heater element 913 may comprise anynumber of devices useful for heating an object such as the chambers ofthe dispenser assembly 902, such as a fluid heat exchanger with tubingin heat exchange contact with the chambers. In a preferred example, butnot as a limitation, electrical resistance-type heaters comprisingcoils, plates, and the like are utilized as part of the heater element913. Preferably, in this embodiment, the resistive portions of theheater element 913 would be formed into a shape that conforms to theshape of the exterior portion of the chambers of the dispenser assembly902 to provide efficient heat transfer but preferably also allow forinsertion and removal of the chambers of the dispenser assembly 902.During operation of the separation system 900, the temperaturecontroller 904 is configured to receive an operating temperature range,to receive and process temperature feedback signals from the temperaturesensor 908, and in response, to selectively operate the heater element913 to first raise the temperature of the chambers of the dispenserassembly 902 to a temperature within the operating temperature range andto second maintain the sensed temperature within the operating range.

For example, a desired operating range for activating a gel ormanipulating other cellular components and their reactions ontothemselves or with agents may be provided as a set point temperature (ordesired activation temperature) with a tolerance provided on either sideof this set point temperature. The temperature controller 904, in thisexample, may operate the heater element 913 to raise the temperature ofthe chambers of the dispenser assembly 902 to a temperature above theset point temperature but below the upper tolerance temperature at whichpoint the heater element 913 may be shut off by the temperaturecontroller 904. When the temperature sensed by the temperature sensor908 drops below the set point temperature but above the lower tolerancetemperature, the temperature controller 904 operates the heater element913 to again raise the sensed temperature to above the set pointtemperature but below the upper tolerance temperature. In this manner,the temperature controller 904 effectively maintains the temperature ofthe chambers in the dispenser assembly 902 within a desired activationtemperature range (which, of course, may be a very small range thatapproximates a single set temperature). In one embodiment, thetemperature controller is or operates as a proportional integralderivative (PID) temperature controller to provide enhanced temperaturecontrol with smaller peaks and abrupt changes in the temperatureproduced by the heater element 913. Additionally, the temperaturecontroller 904 may include visual indicators (such as LEDs) to indicatewhen the sensed temperature is within a set operating range and/or audioalarms to indicate when the sensed temperature is outside the setoperating range.

In another embodiment, the heater element 913 is configured to operateat more than one setting such that it may be operated throughoutoperation of the processing system 900 and is not shut off. For example,the heater element 913 may have a lower setting designed to maintain thechambers of the dispenser assembly 902 at the lower end of the operatingrange (e.g., acceptable activation temperature range) with highersettings that provide heating that brings the chambers up to highertemperatures within the set operating range. In another embodiment, theheater element 913 is configured to heat up at selectable rates (e.g.,change in temperature per unit of time) to enhance the activation orother processing of separated liquids in the dispenser assembly 902.This feature provides the temperature controller 904 with control overthe heating rate provided by the heater element 913.

As discussed previously, the invention provides features that combine toprovide a compact separation system that is particularly adapted foronsite or field use in hospitals and similar environments where space islimited. FIG. 59 illustrates one preferred arrangement of thecentrifugal processing system 900 of FIG. 58 that provides a compactprofile or footprint while facilitating the inclusion of a temperaturecontrol system. An enclosure 916 is included as part of the temperaturecontrol system to provide structural support and protection for thecomponents of the temperature control system. The enclosure 916 may befabricated from a number of structural materials, such as plastic. Theenclosure 916 supports a heater housing 918 that is configured to allowinsertion and removal of the chambers and other elements of thedispenser assembly 902. The heater housing 918 has a wall that containsthe heater element 913 (not shown in FIG. 59) which is connected viacontrol line 914 to the temperature controller 904. The temperaturesensor 908 (not shown in FIG. 59) is also positioned within the heaterhousing 918, and as discussed with reference to FIG. 58, is positionedrelative to the chambers of the dispenser assembly 902 to sense thetemperature of the chambers, and the contained fluid, during operationof the system 900. A temperature feedback signal is transmitted by thetemperature sensor 908 over line 910 to temperature controller 904,which responds by selectively operating the heater element 913 tomaintain the temperature within the heater housing 918 within a selectedoperating range.

Because the separation system 900 includes temperature sensitivecomponents, such as the controller 850, the temperature control systempreferably is configured to monitor and control the temperature withinthe enclosure 916. As illustrated, a temperature sensor 920 is includedto sense the ambient temperature within the enclosure 916 and totransmit a feedback signal over line 922 to temperature controller 904.An air inlet 930, such as a louver, is provided in the enclosure 916 toallow air, A_(IN), to be drawn into and through the enclosure 916 toremove heated air and maintain the temperature within the enclosure 916at an acceptable ambient temperature. To circulate the cooling air, afan 934 is provided to pull the air, A_(IN), into the enclosure 916 andto discharge hotter air, A_(OUT), out of the enclosure 916. The fan 934is selectively operable by the temperature controller 904 via controlsignals over line 938. The size or rating of the fan 934 may vary inembodiments of the invention and is preferably selected based on thevolume of the enclosure 916, the components positioned within theenclosure 916 (e.g., the quantity of heat generated by the separationsystem 900 components), the desired ambient temperature for theenclosure 916, and other cooling design factors.

In an alternate embodiment of the present invention a dispenser 902, asshown in FIG. 60, is provided, for manipulating the cellular fractionwhich has been isolated and collected via outlet lumen 232. In general,the present invention relates to a dispenser 902 which allows for amanual or automated manipulation of a two-phase method for forming anautologous platelet gel 970 composition wherein all of the bloodcomponents for the platelet gel 970 are derived from a patient to whomthe platelet gel 970 will be applied.

The methods of the present invention for preparing an autologousplatelet gel 970 composition, discussed in further detail below, arerepresented in the flow diagrams depicted in FIGS. 61-63. As discussedpreviously, the methods of the present invention begins by forminganticoagulated whole blood 396 which is achieved by collecting apatient's whole blood 394 in a source container 398 having ananticoagulation agent, such as sodium citrate (citrate) or heparin.Preferably, the whole blood 394 is collected and mixed with a 3.8%solution of sodium citrate (referred to herein as “citrate collectionmedium”) specifically in a 9:1 ratio of blood to citrate collectionmedium. A 3.8% solution of sodium citrate is prepared by adding 3.8grams of sodium citrate per 100 ml of water. While a 3.8% sodium citratecollection medium is that which is frequently used to collect andpreserve blood, the person skilled in this art will recognize that theratio of sodium citrate to whole blood could be in the range of about10.9-12.9% mMOL, final concentration.

First, as discussed in detail previously and depicted in FIG. 61,platelet rich plasma 260 and/or platelet poor plasma 262 are formed bycentrifuging a quantity of anticoagulated whole blood 396 that waspreviously drawn from the patient. The platelet rich plasma 260 is firstdrawn from the centrifuge bag 226 and into collection chamber 400.Collection chamber 400 is preferably a syringe, but any container thatwill not contact activate the collected fraction is acceptable. Theplatelet rich plasma 260 can be pumped via outlet pump 803 (FIG. 53)into a collection chamber 400 or the desired fraction can be drawndirectly into dispenser 902.

In the preferred embodiment, depicted in FIG. 62 according to route 951,the platelet rich plasma 260, in centrifuge bag 226, is divided into twoportions and stored in vessels 952 and 960. The first portion isapproximately ¼ to ½ of the total volume of platelet rich plasma 260 andis utilized in phase-one to prepare the thrombin, while the secondportion of platelet rich plasma 260 is utilized in phase-two vessel 960.Once the platelet rich plasma 260 or alternatively the platelet poorplasma 262 (shown in FIG. 61) is obtained, the preferred methods toobtain thrombin and then produce the platelet gel compositions in anexpedited manner, that is, in less than three minutes, are detaileddiagrammatically in routes 951 or 981, shown in FIGS. 62 and 63,respectively and discussed in detail below. If, however, a longerclotting time, that is, in a range of two to eight minutes, is desirousthe method to obtain the platelet gel composition of the presentinvention can proceed along the routes 971 and 987, which are alsodetailed diagrammatically in FIGS. 63 and 63, respectively and discussedin detail below.

Phase-one according to the preferred embodiment (FIG. 62) begins byrestoring the clot-forming process. To accomplish this, an agent(restoration agent) capable of reversing the effects of theanticoagulation agent is added back into the first portion of theplatelet rich plasma 260 stored in vessel 952. Preferably, therestoration agent can be vessel 952 itself or the restoration agent iscontained within vessel 952 prior to the introduction of platelet richplasma 260; however, the restoration agent may also be introduced later.It is furthermore preferable that the contact activator be a materialsuch as but not limited to glass wool 953 or silica, aluminum,diatomaceous earth, kaolin, etc., or non-wettable surfaces such asplastic, siliconized glass, etc. Chemical activators, such as kaolin,can also be used to speed up the clotting time; however, theirsubsequent removal would also be necessary. In the preferred embodiment,a plastic syringe is the preferred container used to collect the desiredfraction. In the presently preferred embodiment of the invention, thereversal of the anticoagulant is accomplished using calcium chloride.However, any substance which is known or found to be functionallyequivalent to calcium chloride, such as, calcium gluconate or calciumcarbonate, in restoring the coagulation activity of citrated blood maybe used in the practice of the present invention. Thus, although calciumchloride is the presently preferred calcium salt for use in theinvention, any calcium salt which functions in a similar manner tocalcium chloride may be used in the invention. Similarly, although manyblood coagulation reactions are currently believed to require calciumions as cofactors, any substance which is known or subsequently found tobe functionally equivalent to calcium in facilitating these coagulationreactions may be used, either individually or in combination withcalcium, in the practice of the present invention. If theanticoagulation agent used was heparin, then heparinase or any othersuitable anticoagulant reversing compound would be used to reverse theeffect of the anticoagulation agent. The concentration of therestoration agent used to reverse the anticoagulation will depend inpart, upon the concentration of the anticoagulation agent in theplatelet rich plasma 260 and the stoichiometry of the chelating andcoagulation reactions. However, the concentration of the restorationagent used to reverse the anticoagulation must be sufficient to achieveclot formation.

Upon restoration of the platelet rich plasma 260 as shown in FIG. 62, aclot 954 will naturally form. The resulting clot 954 is then trituratedby squeezing the clot 954 through glass wool 953 which serves not onlyas a contact activator but also as a filter, thus expressing thrombin955. Alternatively, or in addition a filter 958 having a large micronpore size thereby allowing the removal of clot debris and any activatoror solids that are present. Filter 958 is positioned at the outlet 956of vessel 952. In the preferred embodiment, the thrombin 955 is thenmixed with the second portion of platelet rich plasma (PRP) 260contained within vessel 960 to form the platelet gel composition 970 ofthe present invention in less than three minutes and in quantitiessufficient for clinical use.

Other additives can be added to the above-described process to increasethe concentration of thrombin formed by the intrinsic pathway or theextrinsic pathway.

As discussed in detail above, restoring the clotting cascade function ofcitrated plasma by addition of calcium chloride and exposure to anactivating agent such as glass wool can generate autologous thrombin.The yield of autologous thrombin by this method however, may be low dueto incomplete conversion of prothrombin and the inactivation ofgenerated thrombin by fibrin and antithrombin III. The addition ofmodifying agents, such as epsilon aminocaproic acid, to the plasma mayimprove the yield by reducing the amount of thrombin neutralization. Thegreatest improvement in thrombin yield, however, will be achieved byproviding a thromboplastic material upon which the necessary clottingfactors will assemble to maximize the rate of prothrombin conversion.The activated platelet membrane provides such a stimulant surface andalso enriches the necessary factor V activity by secreting additionalfactor V during platelet degranulation. The addition of exogenouslipoprotein and/or thromboplastic material to the plasma environment mayalso serve to maximize the thrombin generation by activation of bothintrinsic and extrinsic clotting cascades. Additional amplification ofautologous thrombin generation may also be attained by pretreatment ofPRP and/or PPP to block or remove both antithrombin-III and fibrinogenprior to conversion of prothrombin to thrombin. Such modification may beattained by use of appropriate adsorptive agents, antibodies orprecipitating reagents.

In an alternative embodiment, thrombin 955 is mixed with the plateletpoor plasma 262 of phase-two thereby forming the autologous platelet gelcomposition 972 of the present invention in less than three minutes.

A third embodiment of the present invention, route 971, shown in FIG.62, contemplates collecting the original quantity of platelet richplasma (PRP) 260 derived from the anticoagulated whole blood 396 in acontainer, having a wettable surface, such as glass. The platelet richplasma 260 is then recalcified and the platelet gel composition 974forms. The desired platelet gel composition 974 will requireapproximately two to eight minutes to form as opposed to less than athree minute formation as was described in the preferred embodiment.

In the fourth embodiment depicted diagrammatically by route 981 in FIG.63, the platelet poor plasma 262, rather then the platelet rich plasma260, is divided into two portions, as discussed previously in thepreferred embodiment. The first portion, used in phase-one, which isapproximately ¼ to ½ the original volume is stored in a vessel 952having a wettable surface, then the restoration agent, preferablycalcium chloride, is added directly to the platelet poor plasma 262.Surface activation of the restored platelet poor plasma 262 occurs asresult of the vessel's surface and/or the glass wool 953 or othersurface or chemical activators and a clot 962 thus forms. The resultingclot 962 is triturated, as described previously, and the thrombin 955 iscollected. Thrombin 955 is then mixed with the platelet rich plasma 260of phase-two thereby forming the platelet gel sealant composition 973.

In the fifth embodiment, thrombin 955 is mixed with the platelet poorplasma 262 of phase-two thereby forming the platelet gel composition 975in less than three minutes.

The sixth embodiment follows route 987, shown in FIG. 63 wherein theoriginal quantity of platelet poor plasma 262 is collected in acontainer having a wettable surface, such as glass. The platelet poorplasma 262 is then recalcified and the platelet gel composition forms.

The tensile strength of the platelet gel compositions of the presentinvention can be effected by the addition of calcium ions. Consequently,if a stronger bioadhesive sealant composition is desired using themethods discussed above and disclosed in routes 951 and 981, in FIGS. 62and 62, respectively, more calcium ions may be added at the time theserum is introduced into the platelet rich plasma 260 or the plateletpoor plasma 262. Alternatively, if the method of preparing the plateletgel compositions follows routes 971 and 987, depicted in FIGS. 62 and63, respectively, then calcium ions may be introduced directly into theplatelet rich plasma 260 or the platelet poor plasma 262 and theplatelet gel compositions 974 and 976, respectively, will form.

As discussed in further detail below, the time period necessary for theformation of the platelet gel composition of the present invention isdependent on the quantity of serum added. A 1:4, 1:2 and 3:4 ratio ofserum to platelet rich plasma or platelet poor plasma results in theformation of the platelet gel composition in approximately 90, 55 and 30seconds, respectively. Furthermore, due to the fact that thrombin isautocatalytic, it is important that the serum be used within five hoursof preparation, preferably within two hours and ideally immediately.Alternatively, the serum can be chilled or frozen indefinitely.

The platelet gel compositions of this invention may be used for sealinga surgical wound by applying to the wound a suitable amount plateletrich plasma or platelet poor plasma. Moreover, due to the fact that theplatelet gel compositions of the present invention have been preparedsolely from blood components derived from the patient that is to receivethe platelet gel there is a zero probability of introducing a new bloodtransmitted disease to the patient. The methods of the present inventionmay be further modified so that the formed platelet gel compositionfunctions not only as a haemostatic agent, but also as an adjunct towound healing and as a matrix for delivery of drugs and proteins withother biologic activities. For example, it is well known that fibringlue has a great affinity to bind bone fragments which is useful in bonereconstruction, as in plastic surgery or the repair of major bonebreaks. Consequently, in keeping with the autologous nature of theplatelet gel composition of the present invention autologous bone from apatient can be ground or made into powder or the like, and mixed intothe platelet rich plasma obtained in phase-two of the methods of thepresent invention. Autologous thrombin is then mixed in with theplatelet rich plasma and bone fragments in an amount sufficient to allowthe resulting gel to be applied to the desired locale where it congeals.Other materials that may be utilized are, but not limited to, gelatincollagen, degradable polymers, hyaluronic acid, carbohydrates andstarches.

In instances where the desired platelet gel composition of the presentinvention is to further function as a delivery device of drugs andproteins with other biologic activities the method of the presentinvention may be modified as follows. Prior to adding the thrombin,obtained in phase-one, to the platelet rich plasma of phase-two a widevariety of drugs and proteins with other biologic activities may beadded to the platelet rich plasma of phase-two. Examples of the agentsto be added to the platelet rich plasma prior to the addition of theserum include, but are not limited to, analgesic compounds, such asLidocaine, antibacterial compounds, including bactericidal andbacteriostatic compounds, antibiotics (e.g., adriamycin, erythromycin,gentimycin, penicillin, tobramycin), antifungal compounds,anti-inflammatories, antiparasitic compounds, antiviral compounds,anticancer compounds, such as paclitaxol enzymes, enzyme inhibitors,glycoproteins, growth factors (e.g. lymphokines, cytokines), hormones,steroids, glucocorticosteroids, immunomodulators, immunoglobulins,minerals, neuroleptics, proteins, peptides, lipoproteins, tumoricidalcompounds, tumorstatic compounds, toxins and vitamins (e.g., Vitamin A,Vitamin E, Vitamin B, Vitamin C, Vitamin D, or derivatives thereof). Itis also envisioned that selected fragments, portions, derivatives, oranalogues of some or all of the above may be used.

A number of different medical apparatuses and testing methods exist formeasuring and determining coagulation and coagulation-related activitiesof blood. These apparatuses and methods can be used to assist indetermining the optimal formulation of activator, that is, thrombin,calcium and plasma necessary to form the platelet gel composition of thepresent invention. Some of the more successful techniques of evaluatingblood clotting and coagulation are the plunger techniques illustrated byU.S. Pat. No. 4,599,219 to Cooper et al., U.S. Pat. No. 4,752,449 toJackson et al., and U.S. Pat. No. 5,174,961 to Smith, all of which areassigned to the assignee of the present invention, and all of which areincorporated herein by reference.

Automated apparatuses employing the plunger technique for measuring anddetecting coagulation and coagulation-related activities generallycomprise a plunger sensor cartridge or cartridges and a microprocessorcontrolled apparatus into which the cartridge is inserted. The apparatusacts upon the cartridge and the blood sample placed therein to induceand detect the coagulation-related event. The cartridge includes aplurality of test cells, each of which is defined by a tube-like memberhaving an upper reaction chamber where a plunger assembly is located andwhere the analytical test is carried out, and a reagent chamber whichcontains a reagent or reagents. For an activated clotting time (ACT)test, for example, the reagents include an activation reagent toactivate coagulation of the blood. A plug member seals the bottom of areagent chamber. When the test commences, the contents of the reagentchamber are forced into the reaction chamber to be mixed with the sampleof fluid, usually human blood or its components. An actuator, which is apart of the apparatus, lifts the plunger assembly and lowers it, therebyreciprocating the plunger assembly through the pool of fluid in thereaction chamber. The plunger assembly descends by the force of gravity,resisted by a property of the fluid in the reaction chamber, such as itsviscosity. When the property of the sample changes in a predeterminedmanner as a result of the onset or occurrence of a coagulation-relatedactivity, the descent rate of the plunger assembly there through ischanged. Upon a sufficient change in the descent rate, thecoagulation-related activity is detected and indicated by the apparatus.

Using the methods discussed above, cartridges were assembled with serumobtained from either platelet rich plasma or platelet poor plasma, andCaCl₂ in the reagent chambers. Clotting time tests were performed by theautomated process with either platelet rich plasma (PRP) or plateletpoor plasma (PPP) dispersed into the reaction chambers of thecartridges. In the first experiment, the results of which arerepresented in FIG. 64, the amount of serum, the type of plasma fromwhich the serum was derived, and the type of plasma the serum was mixedwith were tested to determine the shortest clotting times. The ratios ofserum to platelet rich plasma or platelet poor plasma that were studiedincluded 1:4, 1:2, and 3:4. In the second set of experiments, theresults of which are represented in FIGS. 66 and 67, the relationshipbetween actual gel time for the platelet gel of the present was comparedto the clotting time in the cartridge, wherein there is a 0, 30, or 60minute delay of adding the serum from its generation. The third set ofexperiments, the results of which are represented in FIGS. 68 and 69,studied the effect of calcium addition on actual gel time versusclotting time in the cartridge. The final set of experiments, theresults of which are represented in FIG. 65, studied the effect ofadding calcium on clotting times.

Although clotting times varied among donors, comparisons of clottingtimes for individual donors show significant effects of the serum toplasma ratio and the calcium concentration. For all donors, the shortestclotting times occurred for the 3:4 ratio, with clotting times that were47% shorter than those for the 1:4 ratio. Although the difference inclotting times for the 3:4 ratio and the 1:2 ratio was not statisticallysignificant, the clotting times were consistently shorter using the 3:4ratio for all donors. These results demonstrate that clotting times maybe shortened by increasing the serum to platelet rich plasma ratio.Similarly, clotting times were significantly affected by the amount ofcalcium added, with the shortest clotting times obtained when no calciumwas added, suggesting that the serum contained levels, of calcium thatwere sufficient to recalcify the citrated platelet rich plasma.Preliminary results from the scale-up experiments suggest thatexperimental clotting times in the cartridges correlate with actual geltimes.

The invention is further illustrated by the following non-limitedexamples. All scientific and technical terms have the meanings asunderstood by one with ordinary skill in the art. The specific exampleswhich follow illustrate the methods in which the bioadhesive sealantcompositions of the present invention may be prepared in a clinicalsetting and are not to be construed as limiting the invention in sphereor scope. The methods may be adapted to variation in order to producecompositions embraced by this invention but not specifically disclosed.Further, variations of the methods to produce the same compositions insomewhat different fashion will be evident to one skilled in the art.

EXAMPLES

The examples herein are meant to exemplify the various aspects ofcarrying out the invention and are not intended to limit the inventionin any way.

Example 1 Preparation of Bioadhesive Sealant Composition Using PlateletRich Plasma and Serum

6 cc's of platelet rich plasma are drawn into receiving chamber 961 and3 cc's per PRP or PPP are drawn into receiving chamber 957 which furthercontains 0.33 cc's of 10% calcium chloride and glass wool. Clotting ofthe contents will occur in two to eight minutes in receiving chamber957. The clot is then squeezed through optional filter 958 and theserum, produced therefrom, is added to the platelet rich plasmacontained in receiving chamber 961 by either mixing or spraying the twocomponents. The platelet rich plasma and the serum will gel withinapproximately three minutes.

The application of the gel using the syringe-type devices 902 asdescribed above maybe less than desirable for may applications.Consequently, in an alternate embodiment the inactive blood componentand thrombin can be mixed and/or injected into a mold having a desiredgeometric shape. The mold may be constructed of a material having awettable surface, such as, but not limited to plastic. In particular,platelet gel of the present invention may be used to temporarily fill,cavities such as but not limited to holes left in the gum from toothextraction and/or holes left in tissue or bone as a result of injury orsurgical procedures. The present invention provides a simpler way ofintroducing platelet gel for specific uses, by providing that theplatelet gel be pre-shaped or molded into a beneficial shape prior tobeing inserted into a cavity. In the case of tooth extraction theplatelet gel may be shaped so as to achieve a basic conical shape. Othershapes such as, but not limited to rods, and rectangles are contemplatedby this invention. The ability to cause the gel to be more, or less,solid and thus malleable may be achieved during the activation sequenceof the gel formation.

The foregoing description is considered as illustrative only of theprinciples of the invention. Furthermore, since numerous modificationsand changes will readily occur to those skilled in the art, it is notdesired to limit the invention to the exact construction and processesshown as described above. Accordingly, all suitable modifications andequivalents may be resorted to falling within the scope of the inventionas defined by the claims which follow.

The foregoing description is considered as illustrative only of theprinciples of the invention. The words “comprise,” “comprising,”“include,” “including,” and “includes” when used in this specificationand in the following claims are intended to specify the presence of oneor more stated features, integers, components, or steps, but they do notpreclude the presence or addition of one or more other features,integers, components, steps, or groups thereof. Furthermore, since anumber of modifications and changes will readily will readily occur tothose skilled in the art, it is not desired to limit the invention tothe exact construction and process shown described above. Accordingly,all suitable modifications and equivalents may be resorted to fallingwithin the scope of the invention as defined by the claims which follow.

1. A platelet gel dispenser, comprising: a first vessel with a chamberand an opening providing an inlet and an outlet to the chamber; a secondvessel with a chamber and an opening providing an inlet and an outlet tothe chamber; means for drawing a platelet rich plasma into the first andsecond chambers and forcing thrombin out of the chamber of the firstvessel and the platelet rich plasma out of the chamber of the secondvessel, the dispensed thrombin and platelet rich plasma combining toform a platelet gel; and a restoration agent and an activation agentpositioned within the chamber of the first vessel in amounts sufficientto allow formation of a clot upon receiving the platelet rich plasmawithin the chamber of the first vessel, wherein the agents arepositioned within the chamber prior to receiving the platelet richplasma, wherein the restoration agent at least partially reverseseffects of an anticoagulation agent in the received platelet rich plasmaand wherein the activation agent activates the platelet rich plasma toform the clot.
 2. The platelet gel dispenser of claim 1, furtherincluding a modifying agent positioned within the chamber of the firstvessel in an amount sufficient to increase the formation of thrombin inthe chamber of the first vessel.
 3. The platelet gel dispenser of claim2, wherein the modifying agent is epsilon aminocaproic acid.
 4. Theplatelet gel dispenser of claim 2, wherein the modifying agent is athromboplastic material.
 5. The platelet gel dispenser of claim 2,wherein the modifying agent is an exogenous lipoprotein.
 6. The plateletgel dispenser of claim 1, further including cells positioned within thechamber of the second vessel.
 7. The platelet gel dispenser of claim 6,wherein the cells are bone cells.
 8. The platelet gel dispenser of claim1, further including a biological agent positioned within the chamber ofthe second vessel.
 9. The platelet gel dispenser of claim 8, wherein thebiological agent is selected from the group consisting of: analgesiccompounds, antibacterial compounds, bactericidal compounds,bacteriostatic compounds, antibiotics, antifungal compounds,anti-inflammatories, antiparasitic compounds, antiviral compounds,anticancer compounds, enzymes, enzyme inhibitors, glycoproteins, growthfactors, hormones, steroids, glucocorticosteroids, immunomodulators,immunoglobulins, minerals, neuroleptics, proteins, peptides,lipoproteins, tumoricidal compounds, tumorstatic compounds, toxins,vitamins and drugs.
 10. The platelet gel dispenser of claim 1, whereinthe drawing and forcing means includes a first liquid line connected tothe opening of the first vessel and a second liquid line connected tothe opening of the second vessel and valving means for selectingconcurrent flow to the first and second liquid lines or non-concurrentflow to the first and second liquid lines.
 11. The platelet geldispenser of claim 10, wherein the drawing and forcing means includes afirst plunger positioned in the chamber of the first vessel and a secondplunger positioned in the chamber of the second vessel.
 12. The plateletgel dispenser of claim 1, wherein the restoration agent is a calciumsalt.
 13. The platelet gel dispenser of claim 12, wherein said calciumsalt is calcium chloride, calcium gluconate, or calcium carbonate. 14.The platelet gel dispenser of claim 1, including a filter positionedoutside the chamber of the first vessel in fluid communication with theopening of the first vessel with a pore size selected to filter thrombinfrom the clot.
 15. The platelet gel dispenser of claim 1, wherein theactivation agent is glass wool, silica aluminum, diatomaceous earth,kaolin, plastic, siliconized glass or a chemical activator.
 16. Theplatelet gel dispenser of claim 1, wherein the chamber of the secondvessel includes a material selected from the group consisting of:gelatin, collagen, degradable polymers, hyaluronic acid, carbohydratesand starches.
 17. A platelet gel dispenser adapted for preparing anddispensing an autologous platelet gel composition, comprising: a firstvessel for storing a first volume platelet rich plasma; a second vesselfor storing a second volume of platelet rich plasma; a lumen assemblyconnected to an opening in the first vessel and to an opening in thesecond vessel, whereby a platelet rich plasma can be provided to thefirst and second vessels concurrently or selectively; means for drawinga platelet rich plasma into the first and second chambers and forcingthrombin out of the chamber of the first vessel and the platelet richplasma out of the chamber of the second vessel, the dispensed thrombinand platelet rich plasma combining to form a platelet gel; sufficientamount of a restoration agent and an activation agent contained withinthe first vessel to allow formation of a clot upon receiving theplatelet rich plasma within the first vessel; and a filter elementpositioned within the first vessel, wherein the filter element isconfigured for filtering thrombin from the clot.
 18. The dispenser ofclaim 17, further including a modifying agent positioned within thefirst vessel in an amount sufficient to increase the formation ofthrombin.
 19. The dispenser of claim 18, wherein the modifying agent isepsilon aminocaproic acid.
 20. The dispenser of claim 18, wherein themodifying agent is a thromboplastic material.
 21. The dispenser of claim18, wherein the modifying agent is an exogenous lipoprotein.
 22. Thedispenser of claim 17, further including cells contained within thesecond vessel.
 23. The dispenser of claim 22, wherein the cells are bonecells.
 24. The dispenser of claim 17, wherein the restoration agent is acalcium salt.
 25. The dispenser of claim 24, wherein the calcium salt iscalcium chloride, calcium gluconate, or calcium carbonate.
 26. Thedispenser of claim 17, further including a biological agent containedwithin the second vessel.
 27. The dispenser of claim 26, wherein thebiological agent is selected from the group consisting of: analgesiccompounds, antibacterial compounds, bactericidal compounds,bacteriostatic compounds, antibiotics, antifungal compounds,anti-inflammatories, antiparasitic compounds, antiviral compounds,anticancer compounds, enzymes, enzyme inhibitors, glycoproteins, growthfactors, hormones, steroids, glucocorticosteroids, immunomodulators,immunoglobulins, minerals, neuroleptics, proteins, peptides,lipoproteins, tumoricidal compounds, tumorstatic compounds, toxins,vitamins and drugs.
 28. The dispenser of 17, wherein the filter has apore size selected to filter the thrombin from the clot.
 29. Thedispenser of claim 28, further including a tube connected to theopenings of the first and second vessels via the lumen for combining thethrombin from the first vessel and the platelet rich plasma from thesecond vessel and including a valve for controlling flow from the lumento the combining tube.
 30. The dispenser of claim 17, wherein the filterelement comprises glass wool which also serves as the activation agent.31. The dispenser of claim 17, wherein the first volume is selected fromthe range of about one half the second volume to about equal to thesecond volume.
 32. The dispenser of claim 17, wherein the restorationagent is heparinase.
 33. The dispenser of claim 17, wherein the secondvessel includes a material selected from the group consisting of:gelatin, collagen, degradable polymers, hyaluronic acid, carbohydratesand starches.
 34. A dispenser adapted for preparing and dispensing aplatelet gel composition, comprising: a first vessel for storing a firstvolume of platelet plasma; a second vessel for storing a second volumeof platelet plasma; a lumen assembly connected to an opening in thefirst vessel and to an opening in the second vessel, whereby theplatelet plasma is provided to the first and second vessels concurrentlyor selectively; means for drawing a platelet rich plasma into the firstand second chambers and forcing thrombin out of the chamber of the firstvessel and the platelet rich plasma out of the chamber of the secondvessel, the dispensed thrombin and platelet rich plasma combining toform a platelet gel; a restoration agent and an activation agentpositioned within the first vessel in an amount sufficient to allowformation of a clot within the first vessel; a filter for separatingthrombin from the clot; and a mixing assembly connected to the openingsof the first and second vessels via the lumen assembly for combining thethrombin from the first vessel and the platelet plasma from the secondvessel, the mixing assembly comprising a mixing chamber and a valve forcontrolling flow from the lumen assembly to the mixing chamber.
 35. Thedispenser of claim 34, further including a modifying agent positionedwithin the first vessel in an amount sufficient to increase theformation of thrombin in the first vessel.
 36. The dispenser of claim35, wherein the modifying agent is epsilon aminocaproic acid.
 37. Thedispenser of claim 35, wherein the modifying agent is a thromboplasticmaterial.
 38. The dispenser of claim 35, wherein the modifying agent isan exogenous lipoprotein.
 39. The dispenser of claim 34, wherein therestoration agent is a calcium salt.
 40. The dispenser of claim 39,wherein the calcium salt is calcium chloride, calcium gluconate, orcalcium carbonate.
 41. The dispenser of claim 34, further includingcells positioned within the second vessel.
 42. The dispenser of claim41, wherein the cells are bone cells.
 43. The dispenser of claim 34,further including a biological agent positioned within the secondvessel.
 44. The dispenser of claim 43, wherein the biological agent isselected from the group consisting of: analgesic compounds,antibacterial compounds, bactericidal compounds, bacteriostaticcompounds, antibiotics, antifungal compounds, anti-inflammatories,antiparasitic compounds, antiviral compounds, anticancer compounds,enzymes, enzyme inhibitors, glycoproteins, growth factors, hormones,steroids, glucocorticosteroids, immunomodulators, immunoglobulins,minerals, neuroleptics, proteins, peptides, lipoproteins, tumoricidalcompounds, tumorstatic compounds, toxins, vitamins and drugs.
 45. Thedispenser of claim 34, wherein the filter is positioned within the firstvessel.
 46. The dispenser of claim 34, wherein the filter elementcomprises glass wool which also serves as the activation agent.
 47. Thedispenser of claim 34, wherein the filter has a pore size selected tofilter the thrombin from the clot.
 48. The dispenser of claim 34,wherein the first volume is selected from the range of about one halfthe second volume to about equal to the second volume.
 49. The dispenserof claim 34, wherein the second vessel includes a material selected fromthe group consisting of: gelatin, collagen, degradable polymers,hyaluronic acid, carbohydrates and starches.
 50. The dispenser of claim34, wherein the platelet plasma is a platelet rich plasma.
 51. Thedispenser of claim 34, wherein the platelet plasma is a platelet poorplasma.
 52. The dispenser of claim 34, wherein the first volume ofplatelet plasma is a first volume of platelet rich plasma and the secondvolume of platelet plasma is a second volume of platelet poor plasma.53. The dispenser of claim 34, wherein the first volume of plateletplasma is a first volume of platelet poor plasma and the second volumeof platelet plasma is a second volume of platelet rich plasma.
 54. Asystem for preparing and dispensing a platelet gel composition,comprising: a centrifuge apparatus for separating a platelet plasma froma sample of anticoagulated whole blood; a dispenser comprising a firstvessel for storing a first volume of the platelet plasma and a secondvessel for storing a second volume of the platelet plasma; a lumenassembly connectable to the centrifuge apparatus and to openings in thefirst and second vessels for providing the platelet plasma from thecentrifuge apparatus to the first and second vessels concurrently orselectively; means for drawing the platelet plasma from the centrifugeapparatus through the lumen assembly and into the first and secondvessels; a restoration agent and an activation agent positioned withinthe first vessel in amounts sufficient to allow formation of a clot uponreceiving the platelet plasma within the first vessel; and a filterpositioned within the first vessel for separating thrombin from theclot.
 55. The system of claim 54, further including a modifying agentpositioned within the first vessel in an amount sufficient to increasethe formation of thrombin in the first vessel.
 56. The system of claim55, wherein the modifying agent is epsilon aminocaproic acid.
 57. Thesystem of claim 55, wherein the modifying agent is a thromboplasticmaterial.
 58. The system of claim 55, wherein the modifying agent is anexogenous lipoprotein.
 59. The system of claim 54, wherein therestoration agent is a calcium salt.
 60. The system of claim 59, whereinthe calcium salt is calcium chloride, calcium gluconate, or calciumcarbonate.
 61. The system of claim 54, further including cellspositioned within the second vessel.
 62. The system of claim 61, whereinthe cells are bone cells.
 63. The system of claim 54, further includinga biological agent positioned within the second vessel.
 64. The systemof claim 63, wherein the biological agent is selected from the groupconsisting of: analgesic compounds, antibacterial compounds,bactericidal compounds, bacteriostatic compounds, antibiotics,antifungal compounds, anti-inflammatories, antiparasitic compounds,antiviral compounds, anticancer compounds, enzymes, enzyme inhibitors,glycoproteins, growth factors, hormones, steroids, glucocorticosteroids,immunomodulators, immunoglobulins, minerals, neuroleptics, proteins,peptides, lipoproteins, tumoricidal compounds, tumorstatic compounds,toxins, vitamins and drugs.
 65. The system of claim 54, wherein thedrawing means is a suction means.
 66. The system of claim 54, whereinthe drawing means includes a first plunger positioned in the firstvessel and a second plunger positioned in the second vessel.
 67. Thesystem of claim 54, wherein the restoration agent is heparinase.
 68. Thesystem of claim 54, wherein the filter element comprises glass woolwhich also serves as the activation agent.
 69. The system of claim 54,wherein the filter has a pore size selected to filter the thrombin fromthe clot.
 70. The system of claim 54, wherein the first volume isselected from the range of about one half the second volume to aboutequal to the second volume.
 71. The system of claim 54, wherein thesecond vessel includes a material selected from the group consisting of:gelatin, collagen, degradable polymers, hyaluronic acid, carbohydratesand starches.
 72. The system of claim 54, wherein the platelet plasma isa platelet rich plasma.
 73. The system of claim 54, wherein the plateletplasma is a platelet poor plasma.
 74. The system of claim 54, whereinthe first volume of platelet plasma is a first volume of platelet richplasma and the second volume of platelet plasma is a second volume ofplatelet poor plasma.
 75. The system of claim 54, wherein the firstvolume of platelet plasma is a first volume of platelet poor plasma andthe second volume of platelet plasma is a second volume of platelet richplasma.